High molecular weight major outer membrane protein of moraxella

ABSTRACT

An isolated and purified outer membrane protein of a Moraxella strain, particularly  M. catarrhalis,  having a molecular mass of about 200 kDa, is provided. The about 200 kDa outer membrane protein as well as nucleic acid molecules encoding the same are useful in diagnostic applications and immunogenic compositions, particularly for in vivo administration to a host to confer protection against disease caused by a bacterial pathogen that produces the about 200 kDa outer membrane protein or produces a protein capable of inducing antibodies in a host specifically reactive with the about 200 kDa outer membrane protein.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/478,370, filed Jun. 7, 1995 now U.S. Pat. No. 5,808,824,which itself is a continuation-in-part of U.S. patent application Ser.No. 08/431,718 filed May 1, 1995.

FIELD OF THE INVENTION

The present invention relates to the field of immunology and isparticularly concerned with outer membrane proteins from Moraxella,methods of production thereof, genes encoding such proteins and usesthereof.

BACKGROUND OF THE INVENTION

Otitis media is the most common illness of early childhood withapproximately 70% of all children suffering at least one bout of otitismedia before the age of seven. Chronic otitis media can lead to hearing,speech and cognitive impairment in children. It is caused by bacterialinfection with Streptococcus pneumoniae (approximately 50%), non-typableHaemophilus influenzae (approximately 30%) and Moraxella (Branhamella)catarrhalis (approximately 20%). In the United States alone, treatmentof otitis media costs between one and two billion dollars per year forantibiotics and surgical procedures, such as tonsillectomies,adenoidectomies and insertion of tympanostomy tubes. Because otitismedia occurs at a time in life when language skills are developing at arapid pace, developmental disabilities specifically related to learningand auditory perception have been documented in youngsters with frequentotitis media.

M. catarrhalis mainly colonizes the respiratory tract and ispredominantly a mucosal pathogen. Studies using cultures of middle earfluid obtained by tympanocentesis have shown that M. catarrhalis causesapproximately 20% of cases of otitis media (ref. 1—Throughout thisapplication, various references are referred to in parenthesis to morefully describe the state of the art to which this invention pertains.Full bibliographic information for each citation is found at the end ofthe specification, immediately preceding the claims. The disclosures ofthese references are hereby incorporated by reference into the presentdisclosure).

The incidence of otitis media caused by M. catarrhalis is increasing. Asways of preventing otitis media caused by pneumococcus and non-typableH. influenzae are developed, the relative importance of M. catarrhalisas a cause of otitis media can be expected to further increase.

M. cattarrhalis is also an important cause of lower respiratory tractinfections in adults, particularly in the setting of chronic bronchitisand emphysema (refs. 2, 3, 4, 5, 6, 7, and 8). M. cattarrhalis alsocauses sinusitis in children and adults (refs. 9, 10. 11, 12, and 13)and occasionally causes invasive disease (refs. 14, 15, 16, 17, 18, and19).

Like other Gram-negative bacteria, the outer membrane of M. cattarrhalisconsists of phospholipids, lipopolysaccharide (LPS), and outer membraneproteins (OMPs). Eight of the M. cattarrhalis OMPs have been identifiedas major components. These are designated by letters A to H, beginningwith OMP A which has a molecular mass of 98 kDa to OMP H which has amolecular mass of 21 kDa (ref. 20).

Recently, a high-molecular-weight outer membrane protein of M.cattarrhalis was purified and characterized (ref. 21). The apparentmolecular mass of this protein varies from 350 kDa to 720 kDa as judgedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).This protein appears to be an oligomer of much smaller proteins orsubunits thereof of molecular mass 120 to 140 kDa and is antigenicallyconserved among strains of Moraxella.

A protein molecular mass of about 300 to 400 kDa named UspA was alsoreported to be present on the surface of Moraxella (ref. 22).

M. cattarrhalis infection may lead to serious disease. It would beadvantageous to provide other outer membrane proteins for M.cattarrhalis and genes encoding such proteins for use as antigens inimmunogenic preparations including vaccines, carriers for other antigensand immunogens and the generation of diagnostic reagents.

SUMMARY OF THE INVENTION

The present invention is directed towards the provision of a purifiedand isolated major outer membrane protein of Moraxella catarrhalis andother Moraxella strains, having an apparent molecular mass of about 200kDa, as well as genes encoding the same.

In accordance with one aspect of the invention, there is provided anisolated and purified, outer membrane protein of a Moraxella strainhaving a molecular weight of about 200 kDa, as determined by SDS-PAGE,or a fragment or an analog thereof. The outer membrane protein may besubstantially in its native conformation (so as to have substantiallyretained the characteristic immunogenicity of the outer membrane proteinin the Moraxella strain) and may be isolated from a M. catarrhalisstrain, such as from M. cattarrhalis 4223. Such isolated and purifiedabout 200 kDa outer membrane protein is substantially free from non-200kDa outer membrane proteins, phospholipids and lipopolysaccharide ofMoraxella. The about 200 kDa outer membrane protein is at least about 70wt % pure, preferably at least about 90 wt % pure, and may be in theform of an aqueous solution thereof. Such about 200 kDa outer membraneprotein may have substantially the amino acid composition shown in TableIII and a deduced amino acid sequence as shown in FIG. 6 (SEQ ID No: 3).

The present invention also provides a purified and isolated nucleic acidmolecule encoding an outer membrane protein of a strain of Moraxellahaving a molecular mass of about 200 kDa, as determined by SDS-PAGE, ora fragment or an analog of the outer membrane protein. The proteinencoded by the nucleic acid molecule may comprise a protein containingthe amino acid sequenceNH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-x-Gln-Gly-Ile(SEQ ID No: 5) particularly where X is Lys (SEQ ID No: 8), for Moraxellacatarrhalis strain 4223 or containing the corresponding amino acidsequence from other Moraxella strains.

In a further aspect of the present invention, there is provided apurified and isolated nucleic acid molecule having a sequence selectedfrom the group consisting of (a) a DNA sequence as set out in FIG. 6(SEQ ID Nos: 1 or 2), or the complementary sequence thereto; (b) a DNAsequence encoding an about 200 kDa protein of a strain of Moraxella andcontaining the amino acid sequenceNH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-x-Gln-Gly-Ile(SEQ ID No: 5), particularly where x is Lys (SEQ ID No: 8) or thecomplementary sequence thereto; (c) a DNA sequence encoding the deducedamino acid sequence as set out in FIG. 6 (SEQ ID No: 3) or thecomplementary sequence thereto; and (d) a nucleotide sequence whichhybridizes under stringent conditions to any one of the sequencesdefined in (a), (b) or (c). The nucleic acid preferably defined in (d)has at least about 90% sequence identity with any one of the sequencesdefined in (a), (b) or (c).

The nucleic acid molecules provided herein may be included in a vectoradapted for transformation of a host. The nucleic acid moleculesprovided herein also may be included in an expression vector adapted fortransformation of a host along with expression means operatively coupledto the nucleic acid molecule for expression by the host of the about 200kDa outer membrane protein of a strain of Noraxella or the fragment orthe analog of the outer membrane protein. A transformed host containingthe expression vector is included within the invention, along with arecombinant outer membrane protein or fragment or analog thereofproducible by the transformed host.

The expression means may include a nucleic acid portion encoding aleader sequence for secretion from the host of the outer membraneprotein or the fragment or the analog of the outer membrane protein. Theexpression means may include a nucleic acid portion encoding alipidation signal for expression from the host of a lipidated form ofthe outer membrane protein or the fragment or analog thereof.

The present invention further includes a live vector for delivery of theouter membrane protein of the invention or a fragment or analog thereof,comprising a vector containing the nucleic acid molecule providedherein. The live vector may be selected from the group consisting of E.coli, Salmonella, BCG, adenovirus, poxvirus, vaccinia and poliovirus.

In accordance with a further aspect of the present invention, there isprovided a peptide having no less than six amino acids and no more than150 amino acids and containing an amino acid sequence corresponding to aportion only of the outer membrane protein of the invention, or afragment or analog thereof. The peptide may be one having the amino acidsequenceNH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile(SEQ ID No: 8) for the Moraxella catarrhalis 4223 strain or the aminoacid sequence for the corresponding peptide for other strains ofMoraxella.

The present invention also provides an immunogenic compositioncomprising an immunoeffective amount of an active component, which maybe the outer membrane protein or fragment or analog thereof, nucleicacid molecules, recombinant outer membrane proteins, fragments oranalogs thereof, live vectors, and/or peptides, as provided herein,along with a pharmaceutically acceptable carrier therefor with theactive component producing an immune response when administered to ahost, which may be a primate, particularly a human.

The immunogenic composition may be formulated as a vaccine for in vivoadministration to a host to confer protection against diseases caused bya bacterial pathogen that produces the about 200 kDa outer membraneprotein or produces a protein capable of inducing antibodies in the hostspecifically reactive with the about 200 kDa outer membrane protein. Inparticular, the bacterial pathogen is a strain of Moraxella,particularly M. cattarrhalis.

The immunogenic composition may be formulated as a microparticlecapsule, ISCOM or liposome preparation. The immunogenic composition maybe used in combination with a targeting molecule for delivery tospecific cells of the immune system as to mucosal surfaces. Sometargeting molecules include vitamin B12 and fragments of bacterialtoxins, as described in WO 92/17167 (Biotech Australia Pty. Ltd.) andmonoclonal antibodies, as described in U.S. Pat. No. 5,194,254 (Barberet al). The immunogenic compositions of the invention (includingvaccines) may further comprise at least one other immunogenic orimmunostimulating material and the immunostimulating material may be atleast one adjuvant.

Suitable adjuvants for use in the present invention include, (but arenot limited to) aluminum phosphate, aluminum hydroxide, QS21, Quil A,derivatives and components thereof, ISCOM matrix, calcium phosphate,calcium hydroxide, zinc hydroxide, a glycolipid analog, an octadecylester of an amino acid, a muramyl dipeptide, polyphosphazene, ISCOPREP,DC-chol, DDBA and a lipoprotein. Advantageous combinations of adjuvantsare described in copending U.S. patent application Ser. No. 08/261,194filed Jun. 16, 1994 and 08/483,856, filed Jun. 7, 1995, assigned to theassignee hereof and the disclosures of which is incorporated herein byreference thereto. The invention further includes an antibody specificfor the outer membrane protein provided herein producible by immunizinga host with an immunogenic composition as provided herein.

In a further aspect of the invention, there is provided a method ofgenerating an immune response in a host comprising administering theretoan immuno-effective amount of the immunogenic composition as providedherein. The immune response may be a humoral or a cell-mediated immuneresponse. The immune response may provide protection to the host againstdiseases caused by a bacterial pathogen that produces the about 200 kDaouter membrane protein or produces a protein capable of inducingantibodies in the host specifically reactive with the about 200 kDaouter membrane protein. In particular, the pathogen is a strain ofMoraxella, including M. cattarrhalis. Hosts in which protection againstdisease may be conferred include primates, including humans.

The present invention provides, in an additional aspect thereof, amethod of producing a vaccine comprising administering the immunogeniccomposition provided herein to a test host to determine an amount and afrequency of administration of the active component to confer protectionagainst disease caused by a bacterial pathogen that produces the about200 kDa outer membrane protein or produces a protein capable of inducingantibodies in the host specifically reactive with the about 200 kDaouter membrane protein, and formulating the active component in a formand amount suitable for administration to a treated host in accordancewith said determined amount and frequency of administration. Inparticular, the pathogen is a strain of Moraxella, including M.cattarrhalis. The treated host may be a human.

A further aspect of the present invention provides a method ofdetermining the presence of nucleic acid encoding an outer membraneprotein of a strain of Moraxella having a molecular mass of about 200kDa, as determined by SDS-PAGE, or fragment or analog thereof, in asample, comprising the steps of:

(a) contacting the sample with the nucleic acid molecule provided hereinto produce duplexes comprising the nucleic acid molecule and any saidnucleic acid molecule encoding the outer membrane protein present in thesample and specifically hybridizable therewith; and

(b) determining the production of the duplexes.

In yet a further aspect of the invention, there is provided a method ofdetermining the presence of antibodies specifically reactive with outermembrane protein of a strain of Moraxella having a molecular mass ofabout 200 kDa, in a sample, comprising the steps of:

(a) contacting the sample with the outer membrane protein as providedherein to produce complexes comprising the outer membrane protein andany said antibodies present in the sample specifically reactivetherewith; and

(b) determining production of the complexes.

In a further aspect of the invention, there is also provided a method ofdetermining the presence of an outer membrane protein of a strain ofMoraxella having a molecular mass of about 200 kDa, in a samplecomprising the steps of:

(a) immunizing a subject with the immunogenic composition as providedherein, to produce antibodies specific for the outer membrane protein;

(b) contacting the sample with the antibodies to produce complexescomprising any outer membrane protein present in the sample and saidouter membrane protein specific antibodies; and

(c) determining production of the complexes. The outer membrane proteinmay be part of a Moraxella catarrhalis strain.

The present invention provides, in a yet further aspect, a diagnostickit for determining the presence of nucleic acid encoding an outermembrane protein of a strain of Moraxella having a molecular mass ofabout 200 kDa, as determined by SDS-PAGE, or fragment or analog thereof,in a sample, comprising:

(a) the nucleic acid molecule as provided herein;

(b) means for contacting the nucleic acid with the sample to produceduplexes comprising the nucleic acid molecule and any said nucleic acidpresent in the sample and hybridizable with the nucleic acid molecule;and

(c) means for determining production of the duplexes.

In yet a further aspect of the invention, there is provided a diagnostickit for determining the presence of antibodies in a sample specificallyreactive with the outer membrane protein of a strain of Moraxella havinga molecular mass of about 200 kDa, as determined by SDS-PAGE,comprising:

(a) the outer membrane protein as provided herein;

(b) means for contacting the outer membrane protein with the sample toproduce complexes comprising the outer membrane protein and any saidantibodies present in the sample; and

(c) means for determining production of the complexes.

The invention also provides a diagnostic kit for detecting the presenceof an outer membrane protein of a strain of Moraxella having a molecularmass of about 200 kDa, in a sample, comprising:

(a) an antibody specific for the about 200 kDa outer membrane protein asprovided herein;

(b) means for contacting the antibody with the sample to produce acomplex comprising the outer membrane protein and outermembrane-specific antibody; and

(c) means for determining production of the complex.

In a further aspect of the invention, there is provided a method ofproducing an isolated and purified outer membrane protein of a strain ofMoraxella having a molecular mass of about 200 kDa, as determined bySDS-PAGE, comprising the steps of:

(a) providing a cell mass of the Moraxella strain;

(b) disrupting the cell mass to provide a cell lysate;

(c) fractionating the cell lysate to provide a fraction containing theouter membrane protein substantially free from other cell lysatecomponents, and

(d) recovering said outer membrane protein.

The bacterial strain may be M. cattarrhalis. The cell lysate may befractionated by gel electrophoresis.

In this application, the term “about 200 kDa protein” is used to definea family of outer membrane proteins of Moraxella having a molecular massof between about 160 and about 230 kDa and includes proteins havingvariations in their amino acid sequences including those naturallyoccurring in various strains of Moraxella. The purified and isolated DNAmolecules comprising a gene encoding the about 200 kDa protein of thepresent invention also include those encoding functional analogs of theabout 200 kDa protein. In this application, a first protein is a“functional analog” of a second protein if the first protein isimmunologically related to and/or has the same function as the secondprotein. The functional analog may be, for example, a fragment of theprotein or a substitution, addition, deletion mutant thereof or a fusionwith a second protein.

Advantages of the present invention include:

a method for isolating purified about 200 kDa outer membrane protein ofa Moraxella strain that produces the outer membrane protein, includingM. cattarrhalis;

a gene encoding an about 200 kDa outer membrane protein of M.cattarrhalis;

an isolated and purified about 200 kDa outer membrane protein isolatablefrom a Moraxella strain; and

diagnostic kits and immunological reagents for specific identificationof Moraxella and hosts infected thereby.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show an analysis of Moraxella catarrhalis cell proteinsby SDS-PAGE. The identification of the lanes and the sources of theproteins are given in Example 2 below;

FIG. 2 shows a comparative analysis of cell proteins from a number of M.cattarrhalis strains by SDS-PAGE analysis and shows the variability inthe molecular weight of the about 200 kDa protein in different strainsof Moraxella. The identification of the lanes and the sources of theproteins are given in Example 4 below;

FIG. 3 shows an analysis of isolated and purified about 200 kDa outermembrane protein of M. cattarrhalis by SDS-PAGE;

FIG. 4 shows the specific recognition of about 200 kDa outer membraneprotein by anti-peptide antiserum. The identification of the lanes andantiserum are given in Example 8 below;

FIG. 5 shows restriction maps of clones containing a gene encoding theabout 200 kDa outer membrane protein of M. cattarrhalis. The openreading frame of the about 200 kDa outer membrane protein is indicatedby the shaded box. Restriction sites are Sal: SalI, N: NcoI, B: BglII,K: KpnI, Xh: XhoI, RV: EcoRV.

FIG. 6 shows the nucleotide sequence (SEQ ID No: 1—entire sequence, SEQID No: 2—coding sequence) of the gene encoding the about 200 kDa outermembrane protein of M. cattarrhalis and the deduced amino acid sequence(SEQ ID No: 3—identified GTG start codon, SEQ ID No: 4—putative ATGstart codon). Peptide 1 (SEQ ID No: 9) and Peptide 2 (SEQ ID No: 10) areidentified by underlining;

FIG. 7A is a restriction enzyme map of the gene encoding the about 200kDa outer membrane protein of M. catarrhalis (SEQ ID No: 1) showingsingle cutting restriction enzymes;

FIG. 7B is a restriction enzyme map of the gene encoding about 200 kDaouter membrane protein of M. catarrhalis (SEQ ID No: 1) showing doublecutting restriction enzymes;

FIG. 8 shows the identification of the GTG initiation codon byexpressing the C-terminal truncations of the gene encoding the about 200kDa outer membrane protein of M. cattarrhalis. Restriction sites are N:NcoI, K: KpnI, H: HindIII, Hp: HpaI, RV: EcoRV, Sal: SalI;

FIG. 9 shows the identification of the GTG initiation codon byutilization of anti-sera specific for N-terminal peptides of the about200 kDa outer membrane protein of M. cattarrhalis. Restriction sites areNco: NcoI, K: KpnI, H: HindIII, RV: EcoRV, Sal: SalI;

FIG. 10 shows the recognition of 200 kDa protein by anti peptide sera;

FIG. 11 shows the construction of vectors for the expression of theabout 200 kDa outer membrane protein of M. cattarrhalis from E. coli.Nco: NcoI, Pst: PstI, Pvu: PvuII, Sca: ScaI, Sal: SalI;

FIG. 12 shows the expression of N-terminal truncations of the about 200kDa outer membrane protein of M. cattarrhalis in E. coli using thebacteriophage T7 promoter;

FIG. 13 shows the expression of the about 200 kDa outer membrane proteinof M. cattarrhalis fused with the LacZ-α-peptide in E. coli ; and

FIG. 14 shows the specific identification of M. catarrhalis expressingthe about 200 kDa outer membrane protein by guinea pig anti-200 kDaspecific antiserum in contrast to other bacteria. Identification of thelanes and bacteria appears below.

GENERAL DESCRIPTION OF THE INVENTION

Referring to FIGS. 1A and 1B and FIG. 2, there is illustrated theseparation of a novel outer membrane protein from a variety of strainsof M. cattarrhalis having a molecular mass about 200 kDa. The presenceof this about 200 kDa protein in a variety of M. cattarrhalis strainsand, in particular, the almost-universal presence in strains isolatedfrom patients suffering from otitis media is shown in Table I. FIG. 3shows the isolated and purified outer membrane protein.

Purified protein was eluted from a gel and used to raise antibodies inguinea pigs. The antibodies specifically recognize only strains of M.cattarrhalis which produce the outer membrane protein (Table I below).

Referring to FIG. 4, there is shown the recognition of the about 200 kDaouter membrane protein by antibodies raised in guinea pigs to asynthesized peptide corresponding to an internal fragment of the about200 kDa protein. The synthesized peptide had the amino acid sequenceNH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys (SEQID No: 6).

Referring to FIG. 5, there is shown restriction maps of clonescontaining a gene encoding the about 200 kDa outer membrane protein. InFIG. 5, the open reading frame of the about 200 kDa gene is shown as asolid box and the GTG start codon is indicated. The nucleotide sequence(SEQ ID No: 1 and 2) of the gene encoding the about 200 kDa outermembrane protein is shown in FIG. 6, along with the deduced amino acidsequence (SEQ ID No: 3) of the protein. Restriction enzyme maps of thegene encoding the about 200 kDa protein are shown in FIGS. 7(A) and7(B). The amino acid composition of the about 200 kDa protein is shownin Table III.

In one embodiment of the present invention, the isolated and purifiedabout 200 kDa outer membrane protein as provided herein is useful forgenerating antibodies that can be used to specifically distinguish M.cattarrhalis from other bacterial pathogens that cause otitis media andother diseases. Thus referring to FIG. 14, there is illustrated animmunoblot showing the specific reactivity of a guinea pig monospecificanti-200 kDa outer membrane protein antiserum produced by immunizingmice with the purified about 200 kDa outer membrane protein as providedherein. The bacterial lysates analyzed were as follows:

Lane Bacterium Source 1 Molecular Weight Standard 2 M. catarrhalis 4223middle ear fluid 3 M. catarrhalis RH408 non-clumping variant of strain4223 4 H. influenzae, MinnA strain meningitis isolate 5 non-typable H.influenzae, SB12 strain otitis media isolate 6 non-typable H.influenzae, SB33 strain otitis media isolate 7. S. pneumoniae type 6ATCC 6306 8. S. pneumoniae type 14 ATCC 6314 9. P. aeruginosa 10. E.coli DH5α

The results shown in FIG. 14 clearly show the usefulness of outermembrane-specific antisera as provided herein to distinguish betweenbacterial pathogens that produce diseases with similar clinicalsymptoms.

In accordance with another aspect of the present invention, there isprovided a vaccine against Moraxella, comprising animmunogenically-effective amount of the outer membrane protein asprovided herein and a physiologically-acceptable carrier therefor. Theouter membrane protein provided herein also may be used as a carrierprotein for hapten, polysaccharides or peptides to make a conjugatevaccine against antigenic determinants unrelated to the about 200 kDaouter membrane protein.

The about 200 kDa outer membrane protein provided herein is useful as adiagnostic reagent, as an antigen for the generation of anti-outermembrane protein antibodies, or as an antigen for vaccination againstthe diseases caused by species of Moraxella or for detecting infectionby Moraxella.

In additional embodiments of the present invention, the about 200 kDaouter membrane protein as provided herein may be used as a carriermolecule to prepare chimeric molecules and conjugate vaccines (includingglycoconjugates) against pathogenic bacteria, including encapsulatedbacteria. Thus, for example, glycoconjugates of the present inventionmay be used to confer protection against disease and infection caused byany bacteria having polysaccharide antigens includinglipooligosaccharides (LOS) and polyribosylphosphate (PRP). Suchbacterial pathogens may include, for example, Haemophilus influenzae,Streptococcus pneumoniae, Escherichia coli, Neisseria meningitidis,Salmonella typhi, Streptococcus mutants, Cryptococcus neoformans,Klebsiella, Staphylococcus aureus and Pseudomonas aeruginosa. Particularantigens which can be conjugated to outer membrane protein and methodsto achieve such conjugations are described in published PCT applicationWO 94/12641, assigned to the assignee hereof and the disclosure of whichis hereby incorporated by reference thereto.

In another embodiment, the carrier function of the outer membraneprotein may be used, for example, to induce immunity toward abnormalpolysaccharides of tumor cells, or to produce anti-tumor antibodies thatcan be conjugated to chemotherapeutic or bioactive agents.

The present invention extends to the use of the nucleic acid moleculesand proteins provided herein as a medicament and in the manufacture of amedicament for the treatment of Moraxella infections.

In a particular embodiment of the invention, there is provided arecombinant about 200 kDa outer membrane protein of Moraxella orfragment or analog thereof or a fusion protein producible by atransformed host containing at least a portion of the gene encoding theabout 200 kDa protein. Referring to FIG. 11, there is shown recombinantvectors for the production of such proteins. In FIG. 11, the filledboxes show 1.9 kb and 4.8 kb C-terminal regions of 200 kD protein gene,that were inserted into a vector, pT7-7, under the control of thebacteriophage T7 promoter. The small open boxes show seven N-terminalamino acids from the vector in the same reading frame. The shaded boxshows 5.5 kb C-terminal region of 200 kD protein, which contained ATGcodon very close to the N-terminus. This gene fragment was fused to lacZα peptide gene (shown in filled box) under the control of lacZ promoter.The full-length gene, that starts from GTG, is shown in a hatched box.

Referring to FIG. 12, there is shown the expression of N-terminaltruncations of the about 200 kDa protein in E. coli. E. coli strain,BL21(DE3)/pLysS, carrying plasmid, pKS94, was grown in LB brothcontaining 100 μg/ml ampicillin to the early log phase and then IPTG wasadded. After culturing for 2 more hours, the bacteria were harvested andlysed. The lysates were assayed on Western blot using anti-200 kDprotein guinea pig serum as a first antibody. Other procedures were asin FIG. 5. Lane 1: prestained molecular weight marker, Lane 2:BL21(DE3)/pLysS carrying pT7-7 with an incorrect insert. Lane 3:L21(DE3)/pLysS carrying pKS94.

Referring to FIG. 13, there is shown the expression of fusion proteincomprising the βgalactosidase α peptide and a portion of the about 200kDa protein in E. coli. E. coli strain, DH5α, carried pKS140. Theplasmid pKS140 carried the C-terminal 5.5 kb fragment of 200 kD proteingene after a N-terminal portion of LacZ-α-peptide in the same readingframe. The E.coli strain was grown to the stationary phase, harvestedand then lysed. The lysate was assayed by Western blotting. Lane 1:prestained molecular weight marker, Lane 2: DH5α carrying pKS140 (totalprotein, 0.5 μg), Lane 3: sonicate of M. cattarrhalis, strain 4223(total protein, 10 μg).

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of vaccination, diagnosis, treatment of Moraxella infections, andin the generation of immunological reagents. A further non-limitingdiscussion of such uses is further presented below.

1. Vaccine Preparation and Use

Immunogenic compositions, including those suitable to be used asvaccines, may be prepared from the about 200 kDa outer membrane proteinas disclosed herein, as well as immunological fragments and fusionsthereof, which may be purified from the bacteria or which may beproduced recombinantly. The vaccine elicits an immune response in asubject which produces antibodies, including anti-200 kDa outer membraneprotein antibodies and antibodies that are opsonizing or bactericidal.Should the vaccinated subject be challenged by Moraxella or otherbacteria that produce proteins capable of producing antibodies thatspecifically recognize 200 kDa outer membrane protein, the antibodiesbind to and inactivate the bacterium. Furthermore, opsonizing orbactericidal anti-200 kDa outer membrane protein antibodies may alsoprovide protection by alternative mechanisms.

Immunogenic compositions including vaccines may be prepared asinjectables, as liquid solutions or emulsions. The about 200 kDa outermembrane protein may be mixed with pharmaceutically acceptableexcipients which are compatible with the about 200 kDa outer membraneprotein. Such excipients may include, water, saline, dextrose, glycerol,ethanol, and combinations thereof. The immunogenic compositions andvaccines may further contain auxiliary substances, such as wetting oremulsifying agents, pH buffering agents, or adjuvants to enhance theeffectiveness thereof. Immunogenic compositions and vaccines may beadministered parenterally, by injection subcutaneously orintramuscularly. Alternatively, the immunogenic compositions formedaccording to the present invention, may be formulated and delivered in amanner to evoke an immune response at mucosal surfaces. Thus, theimmunogenic composition may be administered to mucosal surfaces by, forexample, the nasal or oral (intragastric) routes. Alternatively, othermodes of administration including suppositories and oral formulationsmay be desirable. For suppositories, binders and carriers may include,for example, polyalkalene glycols or triglycerides. Oral formulationsmay include normally employed incipients such as, for example,pharmaceutical grades of saccharine, cellulose and magnesium carbonate.These compositions can take the form of solutions, suspensions, tablets,pills, capsules, sustained release formulations or powders and containabout 1 to 95% of the about 200 kDa outer membrane protein. Theimmunogenic preparations and vaccines are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective, protective and immunogenic. The quantity tobe administered depends on the subject to be treated, including, forexample, the capacity of the individual's immune system to synthesizeantibodies, and if needed, to produce a cell-mediated immune response.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges arereadily determinable by one skilled in the art and may be of the orderof micrograms of the about 200 kDa outer membrane protein. Suitableregimes for initial administration and booster doses are also variable,but may include an initial administration followed by subsequentadministrations. The dosage may also depend on the route ofadministration and will vary according to the size of the host.

The immunogenic preparations including vaccines may comprise as theimmunostimulating material a nucleotide vector comprising at least aportion of the gene encoding the about 200 kDa protein, or the at leasta portion of the gene may be used directly for immunization.

The concentration of the about 200 kDa outer membrane antigen in animmunogenic composition according to the invention is in general about 1to 95%. A vaccine which contains antigenic material of only one pathogenis a monovalent vaccine. Vaccines which contain antigenic material ofseveral pathogens are combined vaccines and also belong to the presentinvention. Such combined vaccines contain, for example, material fromvarious pathogens or from various strains of the same pathogen, or fromcombinations of various pathogens.

Immunogenicity can be significantly improved if the antigens areco-administered with adjuvants, commonly used as 0.05 to 0.1 percentsolution in phosphate-buffered saline. Adjuvants enhance theimmunogenicity of an antigen but are not necessarily immunogenicthemselves. Adjuvants may act by retaining the antigen locally near thesite of administration to produce a depot effect facilitating a slow,sustained release of antigen to cells of the immune system. Adjuvantscan also attract cells of the immune system to an antigen depot andstimulate such cells to elicit immune responses.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune responses to, for example, vaccines. Intrinsicadjuvants, such as lipopolysaccharides, normally are the components ofthe killed or attenuated bacteria used as vaccines. Extrinsic adjuvantsare immunomodulators which are typically non-covalently linked toantigens and are formulated to enhance the host immune responses. Thus,adjuvants have been identified that enhance the immune response toantigens delivered parenterally. Some of these adjuvants are toxic,however, and can cause undesirable side-effects, making them unsuitablefor use in humans and many animals. Indeed, only aluminum hydroxide andaluminum phosphate (collectively commonly referred to as alum) areroutinely used as adjuvants in human and veterinary vaccines. Theefficacy of alum in increasing antibody responses to diphtheria andtetanus toxoids is well established and a HBsAg vaccine has beenadjuvanted with alum. While the usefulness of alum is well establishedfor some applications, it has limitations. For example, alum isineffective for influenza vaccination and inconsistently elicits a cellmediated immune response.

A wide range of extrinsic adjuvants can provoke potent immune responsesto antigens. These include saponins complexed to membrane proteinantigens (immune stimulating complexes), pluronic polymers with mineraloil, killed mycobacteria in mineral oil, Freund's complete adjuvant,bacterial products, such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

To efficiently induce humoral immune responses (HIR) and cell-mediatedimmunity (CMI), immunogens are typically emulsified in adjuvants. Manyadjuvants are toxic, inducing granulomas, acute and chronicinflammations (Freund's complete adjuvant) FCA, cytolysis (saponins andPluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPSand MDP). Although FCA is an excellent adjuvant and widely used inresearch, it is not licensed for use in human or veterinary vaccinesbecause of its toxicity.

Desirable characteristics of ideal adjuvants include:

(1) lack of toxicity;

(2) ability to stimulate a long-lasting immune response;

(3) simplicity of manufacture and stability in long-term storage;

(4) ability to elicit both CMI and HIR to antigens administered byvarious routes, if required;

(5) synergy with other adjuvants;

(6) capability of selectively interacting with populations of antigenpresenting cells (APC);

(7) ability to specifically elicit appropriate T_(H)1 or T_(H)2cell-specific immune responses; and

(8) ability to selectively increase appropriate antibody isotype levels(for example, IgA) against antigens.

U.S. Pat. No. 4,855,283 granted to Lockhoff et al on Aug. 8, 1989 whichis incorporated herein by reference thereto, teaches glycolipidanalogues including N-glycosylamides, N-glycosylureas andN-glycosylcarbamates, each of which is substituted in the sugar residueby an amino acid, as immuno-modulators or adjuvants. Thus, Lockhoff etal. (U.S. Pat. No. 4,855,283 and ref. 27) reported that N-glycolipidanalogs displaying structural similarities to the naturally-occurringglycolipids, such as glycosphospholipids and glycoglycerolipids, arecapable of eliciting strong immune responses in both herpes simplexvirus vaccine and pseudorabies virus vaccine. Some glycolipids have beensynthesized from long chain-alkylamines and fatty acids that are linkeddirectly with the sugars through the anomeric carbon atom, to mimic thefunctions of the naturally occurring lipid residues.

U.S. Pat. No. 4,258,029 granted to Moloney, assigned to the assigneehereof and incorporated herein by reference thereto, teaches thatoctadecyl tyrosine hydrochloride (OTH) functioned as an adjuvant whencomplexed with tetanus toxoid and formalin inactivated type I, II andIII poliomyelitis virus vaccine. Also, Nixon-George et al. (ref. 24),reported that octadecyl esters of aromatic amino acids complexed with arecombinant hepatitis B surface antigen, enhanced the host immuneresponses against hepatitis B virus.

Lipidation of synthetic peptides has also been used to increase theirimmunogenicity. Thus, Wiesmuller (ref. 25) describes a peptide with asequence homologous to a foot-and-mouth disease viral protein coupled toan adjuvant tripalmityl-S-glyceryl-cysteinylserylserine, being asynthetic analogue of the N-terminal part of the lipoprotein from Gramnegative bacteria. Furthermore, Deres et al. (ref. 26) reported in vivopriming of virus-specific cytotoxic T lymphocytes with syntheticlipopeptide vaccine which comprised of modified synthetic peptidesderived from influenza virus nucleoprotein by linkage to a lipopeptide,N-palmityl-S-[2,3-bis(palmitylxy)-(2RS)-propyl-[R]-cysteine (TPC).

2. Immunoassays

The about 200 kDa outer membrane protein of the present invention isuseful as an immunogen for the generation of anti-200 kDa outer membraneprotein antibodies, as an antigen in immunoassays includingenzyme-linked immunosorbent assays (ELISA), RIAs and other non-enzymelinked antibody binding assays or procedures known in the art for thedetection of anti-bacterial, anti-Moraxella, and anti-200 kDa outermembrane protein antibodies. In ELISA assays, the about 200 kDa outermembrane protein is immobilized onto a selected surface, for example, asurface capable of binding proteins such as the wells of a polystyrenemicrotiter plate. After washing to remove incompletely adsorbed about200 kDa outer membrane protein, a nonspecific protein such as a solutionof bovine serum albumin (BSA) that is known to be antigenically neutralwith regard to the test sample may be bound to the selected surface.This allows for, blocking of nonspecific adsorption sites on theimmobilizing surface and thus reduces the background caused bynonspecific bindings of antisera onto the surface.

The immobilizing surface is then contacted with a sample, such asclinical or biological materials, to be tested in a manner conducive toimmune complex (antigen/antibody) formation. This may include dilutingthe sample with diluents, such as solutions of BSA, bovine gammaglobulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sampleis then allowed to incubate for from 2 to 4 hours, at temperatures suchas of the order of about 20° to 37° C. Following incubation, thesample-contacted surface is washed to remove non-immunocomplexedmaterial. The washing procedure may include washing with a solution,such as PBS/Tween or a borate buffer. Following formation of specificimmunocomplexes between the test sample and the bound about 200 kDaouter membrane protein, and subsequent washing, the occurrence, and evenamount, of immunocomplex formation may be determined by subjecting theimmunocomplex to a second antibody having specificity for the firstantibody. If the test sample is of human origin, the second antibody isan antibody having specificity for human immunoglobulins and in generalIgG. To provide detecting means, the second antibody may have anassociated activity such as an enzymatic activity that will generate,for example, a colour development upon incubating with an appropriatechromogenic substrate. Quantification may then be achieved by measuringthe degree of colour generation using, for example, a visiblespectrophotometer.

3. Use of Sequences as Hybridization Probes

The nucleotide sequences of the present invention, comprising thesequence of the about 200 kDa protein gene, now allow for theidentification and cloning of the about 200 kDa protein gene from anyspecies of Moraxella.

The nucleotide sequences comprising the sequence of the about 200 kDaprotein gene of the present invention are useful for their ability toselectively form duplex molecules with complementary stretches of otherabout 200 kDa protein genes. Depending on the application, a variety ofhybridization conditions may be employed to achieve varying degrees ofselectivity of the probe toward the other genes. For a high degree ofselectivity, relatively stringent conditions are used to form theduplexes, such as low salt and/or high temperature conditions, such asprovided by 0.02 M to 0.15 M NaCl at temperatures of between about 50°C. to 70° C. For some applications, less stringent hybridizationconditions are required such as 0.15 M to 0.9 M salt, at temperaturesranging from between about 20° C. to 55° C. Hybridization conditions canalso be rendered more stringent by the addition of increasing amounts offormamide, to destabilize the hybrid duplex. Thus, particularhybridization conditions can be readily manipulated, and will generallybe a method of choice depending on the desired results. In general,convenient hybridization temperatures in the presence of 50% formamideare: 42° C. for a probe which is 95 to 100% homologous to the targetfragment, 37° C. for 90 to 95% homology and 32° C. for 85 to 90%homology.

In a clinical diagnostic embodiment, the nucleic acid sequences of theabout 200 kDa protein genes of the present invention may be used incombination with an appropriate means, such as a label, for determininghybridization. A wide variety of appropriate indicator means are knownin the art, including radioactive, enzymatic or other ligands, such asavidin/biotin and digoxigenin-labelling, which are capable of providinga detectable signal. In some diagnostic embodiments, an enzyme tag suchas urease, alkaline phosphatase or peroxidase, instead of a radioactivetag may be used. In the case of enzyme tags, colorimetric indicatorsubstrates are known which can be employed to provide a means visible tothe human eye or spectrophotometrically, to identify specifichybridization with samples containing about 200 kDa protein genesequences.

The nucleic acid sequences of the about 200 kDa protein genes of thepresent invention are useful as hybridization probes in solutionhybridizations and in embodiments employing solid-phase procedures. Inembodiments involving solid-phase procedures, the test DNA (or RNA) fromsamples, such as clinical samples, including exudates, body fluids (e.g., serum, amniotic fluid, middle ear effusion, sputum, bronchoalveolarlavage fluid) or even tissues, is adsorbed or otherwise affixed to aselected matrix or surface. The fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes comprisingthe nucleic acid sequences of the about 200 kDa protein encoding genesor fragments or analogs thereof of the present invention under desiredconditions. The selected conditions will depend on the particularcircumstances based on the particular criteria required depending on,for example, the G+C contents, type of target nucleic acid, source ofnucleic acid, size of hybridization probe etc. Following washing of thehybridization surface so as to remove non-specifically bound probemolecules, specific hybridization is detected, or even quantified, bymeans of the label. It is preferred to select nucleic acid sequenceportions which are conserved among species of Moraxella. The selectedprobe may be at least 18 bp and may be in the range of about 30 to 90bp.

4. Expression of the About 200 kDa Protein Gene

Plasmid vectors containing replicon and control sequences which arederived from species compatible with the host cell may be used for theexpression of the genes encoding the about 200 kDa protein in expressionsystems. The vector ordinarily carries a replication site, as well asmarking sequences which are capable of providing phenotypic selection intransformed cells. For example, E. coli may be transformed using pBR322which contains genes for ampicillin and tetracycline resistance and thusprovides easy means for identifying transformed cells. The plasmids orphage, must also contain, or be modified to contain, promoters which canbe used by the host cell for expression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host can be used as a transforming vectorin connection with these hosts. For example, the phage in lambda GEM™-11may be utilized in making recombinant phage vectors which can be used totransform host cells, such as E. coli LE392.

Promoters commonly used in recombinant DNA construction include theβ-lactamase (penicillinase) and lactose promoter systems and othermicrobial promoters, such as the T7 promoter system as described in U.S.Pat. No. 4,952,496. Details concerning the nucleotide sequences ofpromoters are known, enabling a skilled worker to ligate themfunctionally with genes. The particular promoter used will generally bea matter of choice depending upon the desired results. Hosts that areappropriate for expression of the about 200 kDa protein genes,fragments, analogs or variants thereof, may include E. coli, Bacillusspecies, Haemophilus, fungi, yeast, Bordetella, or the baculovirusexpression system may be used.

In accordance with this invention, it is preferred to make the proteinby recombinant methods, particularly when the naturally occurring about200 kDa protein as purified from a culture of a species of Moraxella mayinclude trace amounts of toxic materials or other contaminants. Thisproblem can be avoided by using recombinantly produced protein inheterologous systems which can be isolated from the host in a manner tominimize contaminants in the purified material. Particularly desirablehosts for expression in this regard include Gram positive bacteria whichdo not have LPS and are, therefore, endotoxin free. Such hosts includespecies of Bacillus and may be particularly useful for the production ofnon-pyrogenic about 200 kDa protein, fragments or analogs thereof.

Biological Deposits

Certain plasmids that contain portions of the gene having the openreading frame of the gene encoding the about 200 kDa outer membraneprotein of M. cattarrhalis strain 4223 that are described and referredto herein have been deposited with the America Type Culture Collection(ATCC) located at 12301 Parklawn Drive, Rockville, Md., 20852, U.S.A.,pursuant to the Budapest Treaty and pursuant to 37 CFR 1.808 and priorto the filing of this application. The identifications of the respectiveportions of the gene present in these plasmids are shown in FIG. 5.

Samples of the deposited plasmids will become available to the publicupon grant of a patent based upon this United States patent application.The invention described and claimed herein is not to be limited in scopeby plasmids deposited, since the deposited embodiment is intended onlyas an illustration of the invention. Any equivalent or similar plasmidsthat encode similar or equivalent antigens as described in thisapplication are within the scope of the invention.

Plasmid ATCC Designation Date Deposited pKS47 97,111 April 7, 1995 pKS597,110 April 7, 1995 pKS9 97,114 April 18, 1995

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitations.

Methods of molecular genetics, protein biochemistry, and immunology usedbut not explicitly described in this disclosure and these Examples areamply reported in the scientific literature and are well within theability of those skilled in the art.

Example 1

This Example illustrates the generation of a non-clumping strain (RH408)of M. cattarrhalis.

M. cattarrhalis strain 4223, a clumping strain (a common property ofMoraxella strains), was inoculated into several flasks containing 20 mLof brain heat infusion (BHI) broth, and the cultures were incubated withshaking (170 rpm) overnight at 37° C. Five mL of each overnight culturewere transferred to five individual 1 mL tubes, and were left sittingundisturbed at room temperature for 3 to 8 hours, to allow bacteria tosediment. One hundred μL of the cleared upper phase of each culture wereused to inoculate 25 mL of BHI broth and cultures were incubatedovernight at 37° C., as described above. This passaging was repeated sixtimes, using 25 μL of cleared culture to inoculate 25 mL of BHI for eachovernight culture. Non-clumping bacterial cultures were identified bymeasuring the absorbency A₅₇₈, at intervals over a 3 hour time period,in order to compare the sedimentation rates of the passaged strains tothat of the original M. cattarrhalis strain 4223 culture. Non-clumpingmutants, including M. cattarrhalis RH408, did not aggregate during thethree hour time period. On BHI agar plates, strain RH408 had a colonymorphology typical for all non-clumping strains. Strain RH408 waspreviously deposited in connection of U.S. application Ser. No.08/328,589 at the ATCC under the Budapest Treaty on Dec. 13, 1994 withAccession No. 55637.

Example 2

This Example illustrates the identification of the about 200 kDa outermembrane protein of Moraxella catarrhalis.

M. cattarrhalis strains 4223, RH408, 5191, 8185, M2, M5, ATCC 25240, 3,56, 135, 585 were grown in brain heart infusion (BHI) broth. The culturewas incubated overnight with aeration at 37° C.

M. cattarrhalis cells were sonicated and total protein was determinedusing the BCA assay system (Pierce, Rockford, Ill.). Ten μg of totalprotein were mixed with the SDS-PAGE sample buffer containing 0.3MTris-HCl (pH 8.0), 50% glycerol, 10% SDS, 20% β-mercaptoethanol and0.01% bromophenol blue, boiled for 5 minutes and loaded on each lane ofSDS-PAGE gel (0.75 mm thick, 7.5% acrylamide). The gels were run at 200V for 1 hour. Proteins were visualized by staining gels with a solutioncontaining 0.13% Coomassie brilliant blue, 10% acetic acid and 45%methanol. Excess stain was removed with a destaining solution of 5%ethanol and 7.5% acetic acid.

The various Moraxella proteins separated by this procedure are shown inFIGS. 1A and 1B. The M. catarrhalis strains tested were as follows:

Lane Bacterial Strain Source FIG. 1A 1. Molecular Weight Standards 2. E.coli 3. No sample 4. M. catarrhalis 4223 middle ear fluid 5 M.catarrhalis RIT408 non-clumping variant of 4223 6. M. catarrhalis 5191middle ear fluid 7. M. catarrhalis 8185 nasopharynx 8. M. catarrhalis M2sputum 9. M. catarrhalis M5 sputum 10. M. catarrhalis 25240 ATCC 25240FIG. 1B 1. E. coli 2. No sample 3. Molecular Weight Size Markers 4. M.catarrhalis 4223 middle ear fluid 5 M. catarrhalis RH408 non-clumpingvariant of 4223 6. M. catarrhalis 3 sputum 7. M. catarrhalis 56 sputum8. M. catarrhalis 135 middle ear fluid 9. M. catarrhalis 585 Blood

The about 200 kDa outer membrane protein was clearly seen in all otitismedia strains (M. cattarrhalis 4223, 5191, 135), in one strain isolatedfrom the nasopharynx (8185), and in one strain isolated from sputum(M2). However, the about 200 kDa protein was not detected in threeisolates from sputum (3, 56 and M5) and in one strain with unknownorigin (ATCC 25240). A very narrow band was found in an isolate fromblood of a bacteremia patient (585) and this band was recognized by ananti-200 kDa specific guinea pig serum on an immunoblot. Strain RH408 isa non-clumping spontaneous mutant isolated from strain 4223 (seeExample 1) and was found to not express the about 200 kDa protein.

When gels were run longer, they showed heterogeneity in the apparentmolecular masses of the about 200 kDa outer membrane protein indifferent strains of M. catarrhalis (FIG. 2). In FIG. 2 the strainsanalyzed were as follows:

Lane Strain Source 1. Molecular Weight Size Markers 2. M. catarrhalisH04 middle ear fluid 3 M. catarrhalis H12 middle ear fluid 4. M.catarrhalis PO34 middle ear fluid 5. M. catarrhalis PO51 middle earfluid 6. M. catarrhalis E-07 middle ear fluid 7. M. catarrhalis E-22middle ear fluid 8. M. catarrhalis E-23 middle ear fluid 9. M.catarrhalis RH 4223 middle ear fluid 10. M. catarrhalis RH 408Non-clumping variant of 4223

The strain H12 (lane 3) was a natural isolate from middle ear fluid, butdid not produce the about 200 kDa protein.

There may be at least three different sizes of protein in the about 200kDa range. However, antibodies raised against the about 200 kDa outermembrane protein from one strain of M. cattarrhalis (4223) did recognizeall about 200 kDa proteins tested, present in different strains of M.cattarrhalis. It is possible, however, that in particular immunogeniccompositions, for example, as a vaccine and in particular diagnosticembodiments, that the about 200 kDa outer membrane protein from avariety of M. cattarrhalis isolates (including immunogenically diverseisolates) may be required.

Example 3

This Example illustrates the detection of antibodies specific for theabout 200 kDa outer membrane protein in a serum obtained from aconvalescent patient having recovered from otitis media due to M.cattarrhalis.

After separation by SDS-PAGE, bacterial proteins were transferred frompolyacrylamide gels to prepared PVDF (polyvinylidene fluoride;Millipore) membranes at a constant voltage of 70 V for 1.5 h in a buffersystem consisting of 3 g Tris, 14,4 g glycine and 200 ml methanol perliter at 4° C. Membranes with transferred proteins were blocked withBlocking Reagent (from Boehringer Mannheim) diluted in TBS (0.1M Tris,0.15M Nacl) at room temperature for 30 min. Blots were exposed toconvalescent antiserum diluted 1:500 in Blocking Reagent/TBS with 0.1%Tween 20 for 2 hours at room temperature. This patient had otitis mediaand the M. catarrhalis strain isolated from the patient's ear fluid wasM. cattarrhalis CJ7. Blots were then washed 2 times in BlockingReagent/TBS with Tween at 15 min per wash. The reporter conjugate,horseradish peroxidase (HRP) conjugated to protein G, was diluted 1:4000with Blocking Reagent/TBS with Tween and used to immerse the washedmembranes for 30 min at room temperature. Blots were washed twice asabove, followed by a TBS wash. Bound antibodies were detected using theLumiGlo (Kirkegaard and Perry) chemiluminescent detection system asdescribed by the manufacturer. Treated blots were exposed to X-ray film.Antibodies were detected in this convalescent serum that reacted withthe about 200 kDa outer membrane protein of M. cattarrhalis CJ7. Theseresults indicate that the about 200 kDa outer membrane protein is animmunogenic protein of M. cattarrhalis to which an immune response iselicited during a natural infection by M. catarrhalis.

Example 4

This Example illustrates the isolation and purification of the about 200kDa outer membrane protein.

M. cattarrhalis 4223 cells were harvested by centrifugation at 2,000 rpmfor 10 min and frozen. The frozen cells were thawed, resuspended in 20mM sodium phosphate buffer (pH 7.2) and sonicated until the cells weredisrupted. The frozen-thawed cells were also lysed in 20 mM Tris buffer(pH 8) containing 4% SDS and 0.2 mM EDTA by boiling for 5 min to producea cell lysate. The cell sonicates and cell lysates were suspended in aSDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, boiledfor 5 min and separated by SDS-PAGE on a gel (1.5 mm thick, 7.5%acrylamide). The estimated position of the about 200 kDa protein on thegel was cut out and the protein extracted from the gel by electroelutionusing the same buffer as the SDS-PAGE running buffer. The isolated about200 kDa outer membrane protein was shown to be a homogeneous, singleband by SDS-PAGE as seen in FIG. 3. The samples analyzed in FIG. 3 areas follows:

Lane Sample 1. Molecular Weight Size Markers 2. Isolated and purified200 kDa outer membrane protein

The isolated and purified 200 kDa outer membrane protein of M.cattarrhalis shown in FIG. 3 has a purity of at least 70%. Purifiedabout 200 kDa outer membrane protein preparations of at least 95% couldbe readily achieved.

Example 5

This Example illustrates the immunization of guinea pigs with purifiedabout 200 kDa protein from M. catarrhalis.

Approximately 30 to 40 μg of the about 200 kDa protein, which wasisolated from M. cattarrhalis strain 4223 by electroelution, were mixedwith Freund's complete adjuvant (FCA) and subcutaneously injected intoguinea pigs. After two weeks, the animals were boosted with about thesame amount of the about 200 kDa protein in incomplete Freund's adjuvant(IFA). Two weeks later, blood was collected from the guinea pigs andantisera were obtained.

One antiserum was examined on Western blot for its reactivity with theabout 200 kDa protein present in 54 different strains of M.cattarrhalis, which were isolated in different geographical locationsthroughout the world (Canada, U.S. and Finland) (see Table 1 below). Theabout 200 kDa protein was recognized by the antiserum in all strains, inwhich the presence of the about 200 kDa protein band was detected onSDS-PAGE gels stained with Coomassie Blue. These results indicate thatcommon epitopes of the about 200 kDa protein were present in all M.cattarrhalis strains, which possessed this protein. As stated earlier,this protein is not present in all M. catarrhalis strains, but almostall strains, which were isolated from middle ear fluids from otitismedia patients, did possess this protein (Table 1).

Example 6

This Example illustrates the specific recognition of M. cattarrhalisstrain 4223 with anti-200 kDa protein guinea pig serum by ELISA assay(see Table 2 below).

M. cattarrhalis strains 4223, RH408 (200 kDa protein negative mutant)and H-12 were cultured in 60 mL of BHI broth overnight. E. coli strainBL21 (DE3) was cultured in 60 mL of broth overnight. The cultures weresplit into three tubes and centrifuged. M. cattarrhalis strain 4223 wascentrifuged at 1,500 rpm for 10 min., H-12 at 2,000 rpm for 10 min., andRH408 and E. coli BL21 (DE3) at 3,000 rpm for 15 min. The pellet in onetube was suspended in 20 ml of Dulbecco's phosphate buffered saline(D-PBS) and diluted to 1/500 with coating buffer (0.05Mcarbonate/bicarbonate buffer) pH 9.6. One hundred μL of the bacteriasuspension were placed in each well and incubated for 1 hour at roomtemperature. One hundred μL of 0.2% glutaraldehyde was added to eachwell and incubated at room temperature for 10 min. to fix the cells onthe well. The wells were washed with PBS containing 0.1% Tween 20 and0.1% BSA (washing buffer), and then blocked with PBS containing 0.1% BSAfor 30 min. at room temperature. After washing 5 times for 10 secondswith the washing buffer, serial dilutions of guinea pig antiserum withthe washing buffer were added to the wells and incubation at roomtemperature was continued for 60 min. After washing, goat anti-guineapig IgG conjugated with horseradish peroxidase was added to each well atthe dilution of 1/20,000. After incubation at room temperature for 60minutes, the wells were washed and then color reaction was developedusing 3,3-5,5-tetramethylbenzidene (TMB) and hydrogen peroxide.

The ELISA plate wells were also coated with sonicates containing 10μg/mL of total proteins in the coating buffer, blocked without thefixation process and then assayed as described above.

The results shown in Table 2 indicate that the about 200 kDa outermembrane protein specific guinea pig antiserum specifically recognizesstrains of M. catarrhalis which produce the about 200 kDa protein. Theability of the antiserum to recognize whole cells indicates that theprotein is present on the surface of the bacterial cells.

Example 7

This Example describes the determination of an internal amino acidsequence of the 200 kDa outer membrane protein.

The about 200 kDa outer membrane protein was isolated from M.cattarrhalis 4223 by electroelution as described above. The protein wassubjected to CNBr degradation, the proteolytic digests subjected toSDS-PAGE and transferred onto PVDF membrane. A peptide band migrating ata position corresponding to approximately 40 kDa was cut out from themembrane and its N-terminal amino acid sequence was determined. Inanother experiment, the CNBr degradation products of the about 200 kDaprotein were subjected to a direct determination of N-terminal aminoacid sequencing without separating by SDS-PAGE. Both analyses gave anidentical, N-terminal sequence of 20 amino acids with one unidentifiedamino acid at the 17th position. The internal sequence of the 200 kDaouter membrane protein was:

NH₂-Asn-Val-Lys-Ser-Val-lle-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-X-Gln-Gly-lle(SEQ ID No: 5).

Example 8

This Example describes the immunization of guinea pigs with a peptidecorresponding to an internal fragment of the about 200 kDa outermembrane protein and the analysis of the antiserum generated.

Based upon the determination of the amino acid sequence of an internalfragment of the about 200 kDa outer membrane protein as described above,a 16 amino acid long peptide of sequence:

NH₂-Asn-Val-Lys-Ser-Val-lle-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys (SEQID No: 6)

was synthesized using standard procedures. This 16-mer peptide wasconjugated to KLH using Imject Maleimide Activated KLH (Pierce,Rockford, Ill.) and approximately 500 μg of the conjugate was injectedinto guinea pigs using the same immunization and boosting schedule asdescribed above. The guinea pig anti-serum raised against the 16-merinternal amino acid sequence (SEQ ID No: 6) was examined by immunoblotanalysis and found to specifically recognize 200 kDa outer membraneprotein in cell sonicates of M. cattarrhalis 4223. The results are shownin FIG. 4 and indicate that the anti-peptide guinea pig antiserumspecifically recognizes the 200 kDa protein of M. cattarrhalis 4223. Thesamples analyzed in FIG. 4 were as follows:

Lane Sample Antiserum 1. Molecular Weight Markers 2. Purified 200 kDaouter membrane Anti-200 kDa protein protein 3. M. catarrhalis cellsonicate Anti-peptide 1:5000 4 M. catarrhalis cell sonicate Anti-peptide1:1000 5. M. catarrhalis cell sonicate Anti-peptide 1:500 6. M.catarrhalis cell sonicate Pre-immune serum

The results obtained confirm that the peptide corresponding to SEQ IDNos: 5 and 6 are derived from the 200 kDa outer membrane protein.

Example 9

This Example describes the preparation of a M. catarrhalis genomiclibrary.

Chromosomal DNA was isolated as follows:

An M. cattarrhalis cell pellet was resuspended in 20 mL of Tris-EDTA(TE) buffer, pH 7.5. Pronase (final concentration 500 μg/mL) and SDS(final concentration 1%) were added and the suspension was incubated at37° C. for 2 hours. DNA was isolated by sequential extractions once withphenol, twice with phenol-chloroform (1:1), and once withchloroform-isoamyl alcohol (24:1). Extracted DNA was dialyzed against 1MNaCl at 4° C. for 4 hours. This was followed by dialysis against TEbuffer, pH 7.5, at 4° C. for 48 hours (3 buffer changes). DNA wasethanol precipitated from the dialysate. Large-size DNA was collected byspooling on a glass rod, air dried and dissolved in 3 mL water. Smallscale Sau3A (New England BioLabs) restriction digests of chromosomal DNA(final volume 10 μl) were done to establish conditions required toobtain maximal amounts of chromosomal DNA with a size range of 15-23 kb.Large scale digests were prepared once the optimal digestion conditionswere determined. The large scale digests consisted of 50 μL ofchromosomal DNA (290 μg/mL), 33 μL water, 10 μL Sau3A buffer (NewEngland BioLabs), 1 μL BSA (10 mg/ml, New England BioLabs) and 6.3 μLSau3A (0.04 U/μL), and were incubated at 37° C. for 15 min. Reactionswere stopped by the addition of 10 μL 10× loading buffer (100 mMTris-HCl pH 8, 10 mM EDTA, 0.1% bromophenol blue, 50% glycerol).Digested DNA was applied to 0.5% agarose gels (prepared inTris-acetate-EDTA (TAE)) and separated according to size at 50 V for 6hours. The region of the gel encompassing DNA of size 15-23 kb was cutfrom the gel and placed in dialysis tubing (BRL) with 3 mL of TAE. DNAwas electroeluted from the gel-slice overnight at a field strength of 1V/cm. Electroeluted DNA in TAE was extracted once with phenol, once withphenol-chloroform (1:1), and precipitated with ethanol. The dried DNApellet was dissolved in 5 μL water. Size-fractionated chromosomal DNAwas ligated with BamHI cut EMBL3 arms (Promega) using T4 DNA ligase in afinal volume of 9 μL. The entire ligation reaction was packaged intophage λ using a commercial packaging kit (Amersham) following themanufacturer's protocol.

The packaged DNA library was amplified on solid medium. This wasaccomplished by incubating 0.1 ml E. coli strain NM539 plating cellssuspended in 10 mM MgSO₄ with 15-25 μL of the packaged DNA library at37° C. for 15 minutes. Bacteria with adsorbed phage were plated onto BBLplates (10 g BBL trypticase peptone, 5 g NaCl and 15 g agar per liter)using 3 mL of BBL top-agarose (same as BBL plates except agar replacedwith 0.6% agarose) and plates were incubated overnight at 37° C. Phagewere eluted from the top-agarose by adding 3 mL SM buffer (50 mMTris-HCl, pH 7.5, 8 mM MgSO₄, 100 mM NaCl, 0.01% gelatin) to the platesand leaving them at 4° C. for 7 hours. SM buffer containing phage wascollected from the plates, transferred to a screwcap tube and stored at4° C. over chloroform.

Example 10

This Example describes the cloning of a gene encoding the M.cattarrhalis 200 kDa outer membrane protein.

The M. cattarrhalis genomic library in phage lambda EMBL3 was screenedusing an anti-200 kDa protein guinea pig antiserum. A lambda phage clone8II, which expressed an about 200 kDa protein, was confirmed byimmunoblotting of the phage lysate using the about 200 kDa outermembrane-specific antiserum.

Plate lysate cultures of this recombinant phage were prepared. The DNAwas extracted from the plate lysates using a Wizard Lambda Preps DNAPurification System (Promega Corp, Madison, Wis.) according to themanufacturer's instructions. This phage clone carried a DNA insert ofabout 16 kb in size (the restriction map for which is shown in FIG. 5).The phage DNA was digested with a mixture of the restriction enzymesSailI and XhoI, and separated by agarose gel electrophoresis. Two DNAbands, approximately 5 kb and 11 kb in size, respectively, were cut outfrom the gel and extracted using a Geneclean kit (BIO 101 Inc., LaJolla,Calif.) according to the manufacturer's direction.

The smaller 5 kb fragment was ligated into a plasmid vector, pBluescriptII SK +/− (Stratagene Cloning Systems, LaJolla, Calif.), which had beenpreviously digested with SalI and XhoI, to produce plasmid pKS5. Thelarger 11 kb fragment was ligated into a plasmid vector, pSP72 (PromegaCorp., Madison, Wis.), to produce plasmid pKS9. Both ligated plasmidswere used to transform E. coli, strain DH5α.

The lambda phage DNA was also digested with a mixture of XhoI and KpnIand the approximately 1.2 kb fragment was isolated after agarose gelseparation as described above. This 1.2 kb fragment was ligated into aplasmid vector, pGEM-7Zf(+) (Promega Corp., Madison, Wis.), to produceplasmid pKS47. Restriction maps of the plasmid clones are shown in FIG.5.

Example 11

This Example describes the sequencing of the gene encoding the about 200kDa outer membrane protein of M. catarrhalis.

The gene encoding the about 200 kDa outer membrane protein was sequencedusing an Applied Biosystems sequencer. The one strand of the insert inthe plasmid pKS5, was sequenced after construction of a nested set ofdeletions using a Erase-a-Base system (Promega Corp., Madison, Wis.).The plasmid pKS5 was first digested with XhoI and KpnI, treated withexonuclease III to generate a nested set of deletions in the insert andthen recircularized according to the manufacturer's directions. E. coliDH5α was transformed with a series of plasmids with deletions generatedin this way. Plasmids were isolated from the transformants using aQuiagen midi plasmid isolation kit (Qiagen) and the size of plasmidsexamined by agarose gel electrophoresis after restriction enzymedigestion. The inserts of the plasmids with deletions were sequencedusing a bacteriophage T7 promoter sequence as a primer.

Based upon the sequence, nucleotide primers were synthesized. Using thesynthetic nucleotide primers, sequence gaps, which were not sequenced bythe Erase-a Base system, were determined.

The sequences of the inserts in plasmids pKS47 and pKS71 were determinedfrom both ends using synthetic nucleotide primers. The nucleotidesequence of the gene has an open reading frame of the gene coding forthe about 200 kDa outer membrane protein of M. cattarrhalis as shown inFIG. 6 (SEQ ID No: 2). This sequence included a nucleotide sequence:

5′-AATGTCAAATCAGTCATTAACAAAGAACAAGTAAATGATGCCAATAAAAAGCAAGGCATC-3′ (SEQID No: 7)

which encodes the internal amino acid sequence of the about 200 kDaouter membrane protein (SEQ ID No: 5) determined above. This resultconfirms that the cloned gene has an open reading frame of the genecoding for the about 200 kDa outer membrane protein of M. cattarrhalis.The gene encodes a protein having 1992 amino acids, a calculatedmolecular weight of 204,677 and a calculated amino acid composition asshown in Table III below. The deduced amino acid sequence of the proteinis shown in FIG. 6 (SEQ ID No: 3).

Example 12

This Example describes the identification of the start codon of the geneencoding the about 200 kDa gene of M. cattarrhalis.

To identify the translation start codon and the promoter region of the200 kDa protein gene, a plasmid, pKS80, was constructed from pKS5 andpKS47 (FIG. 5). This construct contained about 250 base pairs of DNAupstream from the ATG. The plasmid, pKS5, was digested with KpnI andXhoI. The digest was separated on 0.8% agarose gel and the about 8 kbDNA fragment was cut out from the gel and extracted. Another plasmid,pKS47, was also digested with the two enzymes and the about 1.1 kb DNAfragment was extracted. The 1.1 kb fragment was ligated to the 8 kbfragment to construct pKS80. Western blots using anti-200 kD proteinguinea pig serum failed to detect 200 kD protein in the lysates of thetransformants carrying pKS80.

To examine if the construct was too long to be expressed in E. coli,three different sizes of C-terminal truncations were constructed, asshown in FIG. 8. First, the whole insert in pKS80 was cut out bydigestion with KpnI and BamHI and then inserted into another vectorplasmid, pGEM7Zf(+) (Promega, Madison Wis.), which had been previouslydigested with the same two enzymes. The resulting plasmid, pKS105, wasfurther digested with either one of the following enzymes, (1) HindIII,(2) HpaI and SmaI or (3) EcoRV, gel-purified and then recircularized toproduce pKS130, pKS136 and pKS144, respectively. Transformants of E.coli, DH5α, with either one of pKS130, pKS136 or pKS144 did not produceany truncated proteins, when examined on Western blots using anti-200 kDprotein guinea pig serum.

Next, to investigate if the start codon was GTG and if the promoterregion was further upstream from the GTG, an about 0.9 kb fragment wascut out from pKS71 using ApaI and KpnI, and ligated into pKS130, pKS136and pKS144, which had been previously digested with ApaI and KpnI. The0.9 kb fragment from pKS71 carried the NcoI-KpnI fragment, whichcontained the possible start codon, GTG, and about 700 bp upstreamregion from the GTG (FIG. 8). The resulting constructs, pKS159, pKS149and pKS155, produced truncated proteins, which were recognized byanti-200 kDa protein guinea pig serum on Western blots. The ApaI andKpnI fragment was also ligated to pKS105, which had no C-terminaltruncation, to produce pKS164. The transformants carrying pKS164produced a full-length 200 kDa protein, which was recognized by the sameantiserum on Western blot. These results show that the 5′-region of thegene containing the GTG codon and its upstream sequence is necessary forexpression of the about 200 kDa protein gene from its own promoter in E.coli, and indicate that a translation start codon of the about 200 kDaprotein gene is GTG.

To confirm that the start codon of the gene is GTG, two peptides weresynthesized, as shown in FIG. 9, according to the deduced amino acidsequence from the nucleotide sequence in FIG. 6. Peptide 1 (SEQ ID No:9) encompasses the 30 amino acids from the GTG start codon. Peptide 2(SEQ ID No: 10) is the next 30 amino acid peptide. The peptides areidentified in FIG. 6 by underlining. Antisera were raised against thesetwo peptides in guinea pigs and antisera were obtained. As seen in FIG.10, antisera raised against these two peptides clearly recognized 200kDa protein from M. catarrhalis, strain 4223, by Western blotting. M.catarrhalis, strain 4223, was sonicated. Proteins in the sonicate wereseparated on a SDS-PAGE gel and transferred to PVDF membrane. Themembrane was cut into strips and treated with either anti-peptide 1 oranti-peptide 2 guinea pig serum as a first antibody. The second antibodywas goat anti-guinea pig IgG conjugated with horse radish peroxidase(Jackson ImmunoResearch Lab. Inc., West Grove, Pa.). The membrane wasfinally treated with CN/DAB substrate (Pierce, Rockford, Ill.) for colordevelopment. Lane 1: prestained molecular weight marker, Lane 2:anti-200 kD protein serum, Lane 3: anti-peptide I serum from guinea pigNo. 1, Lane 4: prebleed serum from guinea pig No. 1, Lane 5:anti-peptide 1 serum from guinea pig No. 2, Lane 6: prebleed serum fromguinea pig No. 2, Lane 7: anti-peptide 2 serum from guinea pig No. 3,Lane 8: prebleed serum from guinea pig No. 3, Lane 9: anti-peptide 2serum from guinea pig No. 4, Lane 10: prebleed serum from guinea pig No.4. The results shown in FIG. 10 indicate that the GTG is the translationstart codon of the gene encoding the about 200 kDa protein.

The coding sequence of the about 200 kDa protein gene, which starts atGTG, is 5976 bp and encodes a protein of 1992 amino acids and acalculated molecular weight of 204,677. The position of the 200 kDaprotein gene is shown in FIG. 5. The sequence between NcoI and SalI andits amino acid translation are shown in FIG. 6. The calculated aminoacid composition of the about 200 kDa protein is shown in Table III.

To construct two different sizes of N-terminal truncation genes underthe control of the T7 promoter (as shown in FIG. 11), a ScaI-SalIfragment, which carried the about 1.9 kb 3′-region of the about 200 kDaprotein gene, was cut out from pKS5, and the PvuII-SalI fragment, whichcarried the about 4.8 kb 3′-region, was cut out from pKS80. The twofragments were ligated into a plasmid, pT7-7, previously digested withSmaI and SalI, to produce pKS94 and pKS91, respectively. These ligationsresulted in fusions of 1.9 kb and 4.8 kb 3′-regions with sevenN-terminal amino acids from the vector. When transformants of an E. colistrain, BL21(DE3)/pLysS, with either pKS94 or pKS91 were induced withIPTG, they produced a large quantity of N-terminally truncated 200 kDaprotein. FIG. 12 shows a Western blot showing the expression of thetruncated protein by one of transformants carrying the pKS94 plasmid.

A LacZ fusion of the 3′- 5.5 kb fragment of the about 200 kDa proteingene, as shown in FIG. 11. The 5.8 kb fragment, which contained the 3′-5.5 kb region of about 200 kDa protein gene, was excised from pKS80 bydigestion with PstI, gel-purified, and then ligated to pGEM5Zf(+)(Promega, Madison, Wis.), previously digested with the same enzyme. TheE. coli DH5α clones, which carried the gene in the same direction andreading frame as the LacZ α peptide, were selected by restriction enzymeanalyses. These clones constitutively expressed the fusion protein, asshown in FIG. 13.

SUMMARY OF THE DISCLOSURE

In summary of the disclosure, the present invention provides an isolatedand purified outer membrane protein of a Moraxella strain, particularlyM. cattarrhalis, having a molecular weight of about 200 kDa as well asisolated and purified DNA molecules encoding the outer membrane protein.The invention also provides analogs, truncations and peptidescorresponding to portions of the outer membrane protein. The protein,DNA sequences, recombinant proteins derived therefrom and peptides areuseful for diagnosis, immunization and the generation of diagnostic andimmunological reagents. Modifications are possible within the scope ofthis invention.

TABLE I Presence of the about 200 kDa outer membrane protein in variousisolates of Moraxella catarrhalis Number of isolates^(1.) Number ofisolates containing the 200 kDa Type of Clinical Isolate Examined outermembrane protein Otitis Media 37  36  Sputum/Expectoration/ 13  6Bronchial Secretion Blood 2 2 Nasopharynx 1 1 Unknown 1 0 ^(1.)Thepresence of the about 200 kDa outer membrane protein was determined byimmunoblot analysis using a monospecific guinea pig anti-200 kDa proteinantiserum.

TABLE II Detection of about 200 kDa outer membrane protein of M.catarrhalis by the monospecific anti-200 kDa outer membrane guinea pigantiserum Strain Sample Reciprocal Reactive Titre 4223 Whole cells notfixed 800 RH408 Whole cells not fixed <200 H12 Whole cells not fixed<200 E. coli BL21 Whole cells not fixed <200 4223 Whole cells fixed 3200RH408 Whole cells fixed 200 H12 Whole cells fixed <200 E. coli BL21Whole cells fixed <200 4223 Sonicate 12,800 RH408 Sonicate 800 H12Sonicate 800 E. coli BL21 Sonicate 200

TABLE III Amino acid composition of the about 200 kDa outer membraneprotein of M. catarrhalis Residue Number Percentage (MW) N - Asparagine196 10.9 T - Threonine 221 10.9 K - Lysine 159 10.0 D - Aspartic Acid147 8.3 A - Alanine 219 7.6 V - Valine 148 7.2 I - Isoleucine 116 6.4S - Serine 150 6.4 G - Glycine 222 6.2 L - Leucine 111 6.1 Q - Glutamine83 5.2 E - Glutamic Acid 55 3.5 F - Phenylalanine 40 2.9 R - Arginine 342.6 Y - Tyrosine 27 2.2 H - Histidine 24 1.6 P - Proline 30 1.4 M -Methionine 7 .4 W - Tryptophan 3 .3 B - Aspartic Acid Asparagine 0 .0C - Cysteine 0 .0

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10 6973 base pairs nucleic acid single linear DNA (genomic) CDS708..6683 1 CCATGGATAT GGGCAGGTGT GCTCGCCTGC CGTATGATGG CGATGACACCCCATTTGCCC 60 CATATCTGTA CGATTTGACA TGTGATATGA TTTAACATGT GACATGATTTAACATTGTTT 120 AATACTGTTG CCATCATTAC CATAATTTAG TAACGCATTT AGTAACGCATTTGTAAAAAT 180 CATTGCGCCC CTTTATGTGT ATCATATGAA TAGAATATTA TGATTGTATCTGATTATTGT 240 ATCAGAATGG TGATGCTATA TGATGATGCC TACGAGTTGA TTTGGGTTAATCACTCTATG 300 ATTTGATATA TTTTGAAACT AATCTATTGA CTTAAATCAC CATATGGTTATAATTTAGCA 360 TAATGGTAGG CTTTTTGTAA AAATCACATC GCAATATTGT TCTACTGTTACTACCATGCT 420 TGAATGACGA TCCCAATCAC CAGATTCATT CAAGTGATGT GTTTGTATACGCACCATTTA 480 CCCTAATTAT TTCAATCAAA TGCCTATGTC AGCATGTATC ATTTTTTTAAGGTAAACCAC 540 CATGAATCAC ATCTATAAAG TCATCTTTAA CAAAGCCACA GGCACATTTATGGCAGTGGC 600 AGAGTACGCC AAATCCCACA GCACGGGGGG GGGGTAGCTG TGCTACAGGGCAAGTTGGCA 660 GTGTATGCAC TCTGAGCTTT GCCCGTATTG CCGCGCTCGC TGTCCTC GTGATC GGT 716 Met Ile Gly 1 GCA ACG CTC AGT GGC AGT GCT TAT GCT CAA AAAAAA GAT ACC AAA CAT 764 Ala Thr Leu Ser Gly Ser Ala Tyr Ala Gln Lys LysAsp Thr Lys His 5 10 15 ATC GCA ATT GGT GAA CAA AAC CAG CCA AGA CGC TCAGGC ACT GCC AAG 812 Ile Ala Ile Gly Glu Gln Asn Gln Pro Arg Arg Ser GlyThr Ala Lys 20 25 30 35 GCG GAC GGT GAT CGA GCC ATT GCT ATT GGT GAA AATGCT AAC GCA CAG 860 Ala Asp Gly Asp Arg Ala Ile Ala Ile Gly Glu Asn AlaAsn Ala Gln 40 45 50 GGC GGT CAA GCC ATC GCC ATC GGT AGT AGT AAT AAA ACTGTC AAT GGA 908 Gly Gly Gln Ala Ile Ala Ile Gly Ser Ser Asn Lys Thr ValAsn Gly 55 60 65 AGC AGT TTG GAT AAG ATA GGT ACC GAT GCT ACG GGT CAA GAGTCC ATC 956 Ser Ser Leu Asp Lys Ile Gly Thr Asp Ala Thr Gly Gln Glu SerIle 70 75 80 GCC ATC GGT GGT GAT GTA AAG GCT AGT GGT GAT GCC TCG ATT GCCATC 1004 Ala Ile Gly Gly Asp Val Lys Ala Ser Gly Asp Ala Ser Ile Ala Ile85 90 95 GGT AGT GAT GAC TTA CAT TTG CTT GAT CAG CAT GGT AAT CCT AAA CAT1052 Gly Ser Asp Asp Leu His Leu Leu Asp Gln His Gly Asn Pro Lys His 100105 110 115 CCG AAA GGT ACT CTG ATT AAC GAT CTT ATT AAC GGC CAT GCA GTATTA 1100 Pro Lys Gly Thr Leu Ile Asn Asp Leu Ile Asn Gly His Ala Val Leu120 125 130 AAA GAA ATA CGA AGC TCA AAG GAT AAT GAT GTA AAA TAT AGA CGCACA 1148 Lys Glu Ile Arg Ser Ser Lys Asp Asn Asp Val Lys Tyr Arg Arg Thr135 140 145 ACC GCA AGC GGA CAC GCC AGT ACT GCA GTG GGA GCC ATG TCA TATGCA 1196 Thr Ala Ser Gly His Ala Ser Thr Ala Val Gly Ala Met Ser Tyr Ala150 155 160 CAG GGT CAT TTT TCC AAC GCC TTT GGT ACA CGG GCA ACA GCT AAAAGT 1244 Gln Gly His Phe Ser Asn Ala Phe Gly Thr Arg Ala Thr Ala Lys Ser165 170 175 GCC TAT TCC TTG GCA GTG GGT CTT GCC GCC ACA GCC GAG GGC CAATCT 1292 Ala Tyr Ser Leu Ala Val Gly Leu Ala Ala Thr Ala Glu Gly Gln Ser180 185 190 195 ACA ATC GCT ATT GGT TCT GAT GCA ACA TCT AGC TCG TTG GGAGCG ATA 1340 Thr Ile Ala Ile Gly Ser Asp Ala Thr Ser Ser Ser Leu Gly AlaIle 200 205 210 GCC CTT GGT GCA GGT ACT CGT GCT CAG CTA CAG GGC AGT ATTGCC CTA 1388 Ala Leu Gly Ala Gly Thr Arg Ala Gln Leu Gln Gly Ser Ile AlaLeu 215 220 225 GGT CAA GGT TCT GTT GTC ACT CAG AGT GAT AAT AAT TCT AGACCG GCC 1436 Gly Gln Gly Ser Val Val Thr Gln Ser Asp Asn Asn Ser Arg ProAla 230 235 240 TAT ACA CCA AAT ACC CAG GCA CTA GAC CCC AAG TTT CAA GCCACC AAT 1484 Tyr Thr Pro Asn Thr Gln Ala Leu Asp Pro Lys Phe Gln Ala ThrAsn 245 250 255 AAT ACG AAG GCG GGT CCA CTT TCC ATT GGT AGT AAC TCT ATCAAA CGT 1532 Asn Thr Lys Ala Gly Pro Leu Ser Ile Gly Ser Asn Ser Ile LysArg 260 265 270 275 AAA ATC ATC AAT GTC GGT GCA GGT GTT AAT AAA ACC GATGCG GTC AAT 1580 Lys Ile Ile Asn Val Gly Ala Gly Val Asn Lys Thr Asp AlaVal Asn 280 285 290 GTG GCA CAG CTA GAA GCG GTG GTG AAG TGG GCT AAG GAGCGT AGA ATT 1628 Val Ala Gln Leu Glu Ala Val Val Lys Trp Ala Lys Glu ArgArg Ile 295 300 305 ACT TTT CAG GGT GAT GAT AAC AGT ACT GAC GTA AAA ATAGGT TTG GAT 1676 Thr Phe Gln Gly Asp Asp Asn Ser Thr Asp Val Lys Ile GlyLeu Asp 310 315 320 AAT ACT TTA ACT ATT AAA GGT GGT GCA GAG ACC AAC GCATTA ACC GAT 1724 Asn Thr Leu Thr Ile Lys Gly Gly Ala Glu Thr Asn Ala LeuThr Asp 325 330 335 AAT AAT ATC GGT GTG GTA AAA GAG GCT GAT AAT AGT GGTCTG AAA GTT 1772 Asn Asn Ile Gly Val Val Lys Glu Ala Asp Asn Ser Gly LeuLys Val 340 345 350 355 AAA CTT GCT AAA ACT TTA AAC AAT CTT ACT GAG GTGAAT ACA ACT ACA 1820 Lys Leu Ala Lys Thr Leu Asn Asn Leu Thr Glu Val AsnThr Thr Thr 360 365 370 TTA AAT GCC ACA ACC ACA GTT AAG GTA GGT AGT AGTAGT AGT ACT ACA 1868 Leu Asn Ala Thr Thr Thr Val Lys Val Gly Ser Ser SerSer Thr Thr 375 380 385 GCT GAA TTA TTG AGT GAT AGT TTA ACC TTT ACC CAGCCC AAT ACA GGC 1916 Ala Glu Leu Leu Ser Asp Ser Leu Thr Phe Thr Gln ProAsn Thr Gly 390 395 400 AGT CAA AGC ACA AGC AAA ACC GTC TAT GGC GTT AATGGG GTG AAG TTT 1964 Ser Gln Ser Thr Ser Lys Thr Val Tyr Gly Val Asn GlyVal Lys Phe 405 410 415 ACT AAT AAT GCA GAA ACA ACA GCA GCA ATC GGC ACTACT CGT ATT ACC 2012 Thr Asn Asn Ala Glu Thr Thr Ala Ala Ile Gly Thr ThrArg Ile Thr 420 425 430 435 AGA GAT AAA ATT GGC TTT GCT CGA GAT GGT GATGTT GAT GAA AAA CAA 2060 Arg Asp Lys Ile Gly Phe Ala Arg Asp Gly Asp ValAsp Glu Lys Gln 440 445 450 GCA CCA TAT TTG GAT AAA AAA CAA CTT AAA GTGGGT AGT GTT GCA ATT 2108 Ala Pro Tyr Leu Asp Lys Lys Gln Leu Lys Val GlySer Val Ala Ile 455 460 465 ACC ATA GAC AAT GGC ATT GAT GCA GGT AAT AAAAAG ATC AGT AAT CTT 2156 Thr Ile Asp Asn Gly Ile Asp Ala Gly Asn Lys LysIle Ser Asn Leu 470 475 480 GCC AAA GGT AGC AGT GCT AAC GAT GCG GTT ACCATC GAA CAG CTC AAA 2204 Ala Lys Gly Ser Ser Ala Asn Asp Ala Val Thr IleGlu Gln Leu Lys 485 490 495 GCC GCC AAG CCT ACT TTA AAC GCA GGC GCT GGCATC AGT GTC ACA CCT 2252 Ala Ala Lys Pro Thr Leu Asn Ala Gly Ala Gly IleSer Val Thr Pro 500 505 510 515 ACT GAA ATA TCA GTT GAT GCT AAG AGT GGCAAT GTT ACC GCC CCA ACT 2300 Thr Glu Ile Ser Val Asp Ala Lys Ser Gly AsnVal Thr Ala Pro Thr 520 525 530 TAC AAC ATT GGC GTG AAA ACC ACC GAG CTTAAC AGT GAT GGC ACT AGT 2348 Tyr Asn Ile Gly Val Lys Thr Thr Glu Leu AsnSer Asp Gly Thr Ser 535 540 545 GAT AAA TTT AGT GTT AAG GGT AGT GGT ACGAAC AAT AGC TTA GTT ACC 2396 Asp Lys Phe Ser Val Lys Gly Ser Gly Thr AsnAsn Ser Leu Val Thr 550 555 560 GCC GAA CAT TTG GCA AGC TAT CTA AAT GAAGTC AAT CGA ACG GCT GAC 2444 Ala Glu His Leu Ala Ser Tyr Leu Asn Glu ValAsn Arg Thr Ala Asp 565 570 575 AGT GCT CTA CAA AGC TTT ACC GTT AAA GAAGAA GAC GAT GAT GAC GCC 2492 Ser Ala Leu Gln Ser Phe Thr Val Lys Glu GluAsp Asp Asp Asp Ala 580 585 590 595 AAC GCT ATC ACC GTG GCT AAA GAT ACGACA AAA AAT GCC GGC GCA GTC 2540 Asn Ala Ile Thr Val Ala Lys Asp Thr ThrLys Asn Ala Gly Ala Val 600 605 610 AGC ATC TTA AAA CTC AAA GGT AAA AACGGT CTA ACG GTT GCT ACC AAA 2588 Ser Ile Leu Lys Leu Lys Gly Lys Asn GlyLeu Thr Val Ala Thr Lys 615 620 625 AAA GAT GGT ACG GTT ACC TTT GGG CTTAGC CAA GAT AGC GGT CTG ACC 2636 Lys Asp Gly Thr Val Thr Phe Gly Leu SerGln Asp Ser Gly Leu Thr 630 635 640 ATT GGC AAA AGC ACC CTA AAC AAC GATGGC TTG ACT GTT AAA GAT ACC 2684 Ile Gly Lys Ser Thr Leu Asn Asn Asp GlyLeu Thr Val Lys Asp Thr 645 650 655 AAC GAA CAA ATC CAA GTC GGT GCT AATGGC ATT AAA TTT ACT AAT GTG 2732 Asn Glu Gln Ile Gln Val Gly Ala Asn GlyIle Lys Phe Thr Asn Val 660 665 670 675 AAT GGT AGT AAT CCA GGT ACT GGCATT GCA AAT ACC GCT CGC ATT ACC 2780 Asn Gly Ser Asn Pro Gly Thr Gly IleAla Asn Thr Ala Arg Ile Thr 680 685 690 AGA GAT AAA ATT GGC TTT GCT GGTTCT GAT GGT GCA GTT GAT ACA AAC 2828 Arg Asp Lys Ile Gly Phe Ala Gly SerAsp Gly Ala Val Asp Thr Asn 695 700 705 AAA CCT TAT CTT GAT CAA GAC AAGCTA CAA GTT GGC AAT GTT AAG ATT 2876 Lys Pro Tyr Leu Asp Gln Asp Lys LeuGln Val Gly Asn Val Lys Ile 710 715 720 ACC AAC ACT GGC ATT AAC GCA GGTGGT AAA GCC ATC ACA GGG CTG TCC 2924 Thr Asn Thr Gly Ile Asn Ala Gly GlyLys Ala Ile Thr Gly Leu Ser 725 730 735 CCA ACA CTG CCT AGC ATT GCC GATCAA AGT AGC CGC AAC ATA GAA CTG 2972 Pro Thr Leu Pro Ser Ile Ala Asp GlnSer Ser Arg Asn Ile Glu Leu 740 745 750 755 GGC AAT ACA ATC CAA GAC AAAGAC AAA TCC AAC GCT GCC AGC ATT AAT 3020 Gly Asn Thr Ile Gln Asp Lys AspLys Ser Asn Ala Ala Ser Ile Asn 760 765 770 GAT ATA TTA AAT ACA GGC TTTAAC CTA AAA AAT AAT AAC AAC CCC ATT 3068 Asp Ile Leu Asn Thr Gly Phe AsnLeu Lys Asn Asn Asn Asn Pro Ile 775 780 785 GAC TTT GTC TCC ACT TAT GACATT GTT GAC TTT GCC AAT GGC AAT GCC 3116 Asp Phe Val Ser Thr Tyr Asp IleVal Asp Phe Ala Asn Gly Asn Ala 790 795 800 ACC ACC GCC ACA GTA ACC CATGAT ACC GCT AAC AAA ACC AGT AAA GTG 3164 Thr Thr Ala Thr Val Thr His AspThr Ala Asn Lys Thr Ser Lys Val 805 810 815 GTA TAT GAT GTG AAT GTG GATGAT ACA ACC ATT CAT CTA ACA GGC ACT 3212 Val Tyr Asp Val Asn Val Asp AspThr Thr Ile His Leu Thr Gly Thr 820 825 830 835 GAT GAC AAT AAA AAA CTTGGC GTC AAA ACC ACC AAA CTG AAC AAA ACA 3260 Asp Asp Asn Lys Lys Leu GlyVal Lys Thr Thr Lys Leu Asn Lys Thr 840 845 850 AGT GCT AAT GGT AAT ACAGCA ACT AAC TTT AAT GTT AAC TCT AGT GAT 3308 Ser Ala Asn Gly Asn Thr AlaThr Asn Phe Asn Val Asn Ser Ser Asp 855 860 865 GAA GAT GCC CTT GTT AACGCC AAA GAC ATC GCC GAA AAT CTA AAC ACC 3356 Glu Asp Ala Leu Val Asn AlaLys Asp Ile Ala Glu Asn Leu Asn Thr 870 875 880 CTA GCC AAG GAA ATT CACACC ACC AAA GGC ACA GCA GAC ACC GCC CTA 3404 Leu Ala Lys Glu Ile His ThrThr Lys Gly Thr Ala Asp Thr Ala Leu 885 890 895 CAA ACC TTT ACC GTT AAAAAG GTA GAT GAA AAT AAT AAT GCT GAT GAC 3452 Gln Thr Phe Thr Val Lys LysVal Asp Glu Asn Asn Asn Ala Asp Asp 900 905 910 915 GCC AAC GCC ATC ACCGTG GGT CAA AAG AAC GCA AAT AAT CAA GTC AAC 3500 Ala Asn Ala Ile Thr ValGly Gln Lys Asn Ala Asn Asn Gln Val Asn 920 925 930 ACC CTA ACA CTC AAAGGT GAA AAC GGT CTT AAT ATT AAA ACC GAC AAA 3548 Thr Leu Thr Leu Lys GlyGlu Asn Gly Leu Asn Ile Lys Thr Asp Lys 935 940 945 AAT GGT ACG GTT ACCTTT GGC ATT AAC ACC ACA AGC GGT CTT AAA GCC 3596 Asn Gly Thr Val Thr PheGly Ile Asn Thr Thr Ser Gly Leu Lys Ala 950 955 960 GGC AAA AGC ACC CTAAAC GAC GGT GGC TTG TCT ATT AAA AAC CCC ACT 3644 Gly Lys Ser Thr Leu AsnAsp Gly Gly Leu Ser Ile Lys Asn Pro Thr 965 970 975 GGT AGC GAA CAA ATCCAA GTC GGT GCT GAT GGC GTG AAG TTT GCC AAG 3692 Gly Ser Glu Gln Ile GlnVal Gly Ala Asp Gly Val Lys Phe Ala Lys 980 985 990 995 GTT AAT AAT AATGGT GTT GTA GGT GCT GGC ATT GAT GGC ACA ACT CGC 3740 Val Asn Asn Asn GlyVal Val Gly Ala Gly Ile Asp Gly Thr Thr Arg 1000 1005 1010 ATT ACC AGAGAT GAA ATT GGC TTT ACT GGG ACT AAT GGC TCA CTT GAT 3788 Ile Thr Arg AspGlu Ile Gly Phe Thr Gly Thr Asn Gly Ser Leu Asp 1015 1020 1025 AAA AGCAAA CCC CAC CTA AGC AAA GAC GGC ATT AAC GCA GGT GGT AAA 3836 Lys Ser LysPro His Leu Ser Lys Asp Gly Ile Asn Ala Gly Gly Lys 1030 1035 1040 AAGATT ACC AAC ATT CAA TCA GGT GAG ATT GCC CAA AAC AGC CAT GAT 3884 Lys IleThr Asn Ile Gln Ser Gly Glu Ile Ala Gln Asn Ser His Asp 1045 1050 1055GCT GTG ACA GGC GGC AAG ATT TAT GAT TTA AAA ACC GAA CTT GAA AAC 3932 AlaVal Thr Gly Gly Lys Ile Tyr Asp Leu Lys Thr Glu Leu Glu Asn 1060 10651070 1075 AAA ATC AGC AGT ACT GCC AAA ACA GCA CAA AAC TCA TTA CAC GAATTC 3980 Lys Ile Ser Ser Thr Ala Lys Thr Ala Gln Asn Ser Leu His Glu Phe1080 1085 1090 TCA GTA GCA GAT GAA CAA GGT AAT AAC TTT ACG GTT AGT AACCCT TAC 4028 Ser Val Ala Asp Glu Gln Gly Asn Asn Phe Thr Val Ser Asn ProTyr 1095 1100 1105 TCC AGT TAT GAC ACC TCA AAG ACC TCT GAT GTC ATC ACCTTT GCA GGT 4076 Ser Ser Tyr Asp Thr Ser Lys Thr Ser Asp Val Ile Thr PheAla Gly 1110 1115 1120 GAA AAC GGC ATT ACC ACC AAG GTA AAT AAA GGT GTGGTG CGT GTG GGC 4124 Glu Asn Gly Ile Thr Thr Lys Val Asn Lys Gly Val ValArg Val Gly 1125 1130 1135 ATT GAC CAA ACC AAA GGC TTA ACC ACG CCT AAGCTG ACC GTG GGT AAT 4172 Ile Asp Gln Thr Lys Gly Leu Thr Thr Pro Lys LeuThr Val Gly Asn 1140 1145 1150 1155 AAT AAT GGC AAA GGC ATT GTC ATT GACAGC CAA AAT GGT CAA AAT ACC 4220 Asn Asn Gly Lys Gly Ile Val Ile Asp SerGln Asn Gly Gln Asn Thr 1160 1165 1170 ATC ACA GGA CTA AGC AAC ACT CTAGCT AAT GTT ACC AAT GAT AAA GGT 4268 Ile Thr Gly Leu Ser Asn Thr Leu AlaAsn Val Thr Asn Asp Lys Gly 1175 1180 1185 AGC GTA CGC ACC ACA GAA CAGGGC AAT ATA ATC AAA GAC GAA GAC AAA 4316 Ser Val Arg Thr Thr Glu Gln GlyAsn Ile Ile Lys Asp Glu Asp Lys 1190 1195 1200 ACC CGT GCC GCC AGC ATTGTT GAT GTG CTA AGC GCA GGC TTT AAC TTG 4364 Thr Arg Ala Ala Ser Ile ValAsp Val Leu Ser Ala Gly Phe Asn Leu 1205 1210 1215 CAA GGC AAT GGT GAAGCG GTT GAC TTT GTC TCC ACT TAT GAC ACC GTC 4412 Gln Gly Asn Gly Glu AlaVal Asp Phe Val Ser Thr Tyr Asp Thr Val 1220 1225 1230 1235 AAC TTT GCCGAT GGC AAT GCC ACC ACC GCT AAG GTG ACC TAT GAT GAC 4460 Asn Phe Ala AspGly Asn Ala Thr Thr Ala Lys Val Thr Tyr Asp Asp 1240 1245 1250 ACA AGCAAA ACC AGT AAA GTG GTC TAT GAT GTC AAT GTG GAT GAT ACA 4508 Thr Ser LysThr Ser Lys Val Val Tyr Asp Val Asn Val Asp Asp Thr 1255 1260 1265 ACCATT GAA GTT AAA GAT AAA AAA CTT GGC GTA AAA ACC ACC ACA TTG 4556 Thr IleGlu Val Lys Asp Lys Lys Leu Gly Val Lys Thr Thr Thr Leu 1270 1275 1280ACC AGT ACT GGC ACA GGT GCT AAT AAA TTT GCC CTA AGC AAT CAA GCT 4604 ThrSer Thr Gly Thr Gly Ala Asn Lys Phe Ala Leu Ser Asn Gln Ala 1285 12901295 ACT GGC GAT GCG CTT GTC AAG GCC AGT GAT ATC GTT GCT CAT CTA AAC4652 Thr Gly Asp Ala Leu Val Lys Ala Ser Asp Ile Val Ala His Leu Asn1300 1305 1310 1315 ACC TTA TCT GGC GAC ATC CAA ACT GCC AAA GGG GCA AGCCAA GCG AAC 4700 Thr Leu Ser Gly Asp Ile Gln Thr Ala Lys Gly Ala Ser GlnAla Asn 1320 1325 1330 AAC TCA GCA GGC TAT GTG GAT GCT GAT GGC AAT AAGGTC ATC TAT GAC 4748 Asn Ser Ala Gly Tyr Val Asp Ala Asp Gly Asn Lys ValIle Tyr Asp 1335 1340 1345 AGT ACC GAT AAC AAG TAC TAT CAA GCC AAA AATGAT GGC ACA GTT GAT 4796 Ser Thr Asp Asn Lys Tyr Tyr Gln Ala Lys Asn AspGly Thr Val Asp 1350 1355 1360 AAA ACC AAA GAA GTT GCC AAA GAC AAA CTGGTC GCC CAA GCC CAA ACC 4844 Lys Thr Lys Glu Val Ala Lys Asp Lys Leu ValAla Gln Ala Gln Thr 1365 1370 1375 CCA GAT GGC ACA TTG GCT CAA ATG AATGTC AAA TCA GTC ATT AAC AAA 4892 Pro Asp Gly Thr Leu Ala Gln Met Asn ValLys Ser Val Ile Asn Lys 1380 1385 1390 1395 GAA CAA GTA AAT GAT GCC AATAAA AAG CAA GGC ATC AAT GAA GAC AAC 4940 Glu Gln Val Asn Asp Ala Asn LysLys Gln Gly Ile Asn Glu Asp Asn 1400 1405 1410 GCC TTT GTT AAA GGA CTTGAA AAA GCC GCT TCT GAT AAC AAA ACC AAA 4988 Ala Phe Val Lys Gly Leu GluLys Ala Ala Ser Asp Asn Lys Thr Lys 1415 1420 1425 AAC GCC GCA GTA ACTGTG GGT GAT TTA AAT GCC GTT GCC CAA ACA CCG 5036 Asn Ala Ala Val Thr ValGly Asp Leu Asn Ala Val Ala Gln Thr Pro 1430 1435 1440 CTG ACC TTT GCAGGG GAT ACA GGC ACA ACG GCT AAA AAA CTG GGC GAG 5084 Leu Thr Phe Ala GlyAsp Thr Gly Thr Thr Ala Lys Lys Leu Gly Glu 1445 1450 1455 ACT TTG ACCATC AAA GGT GGG CAA ACA GAC ACC AAT AAG CTA ACC GAT 5132 Thr Leu Thr IleLys Gly Gly Gln Thr Asp Thr Asn Lys Leu Thr Asp 1460 1465 1470 1475 AATAAC ATC GGT GTG GTA GCA GGT ACT GAT GGC TTC ACT GTC AAA CTT 5180 Asn AsnIle Gly Val Val Ala Gly Thr Asp Gly Phe Thr Val Lys Leu 1480 1485 1490GCC AAA GAC CTA ACC AAT CTT AAC AGC GTT AAT GCA GGT GGC ACC AAA 5228 AlaLys Asp Leu Thr Asn Leu Asn Ser Val Asn Ala Gly Gly Thr Lys 1495 15001505 ATT GAT GAC AAA GGC GTG TCT TTT GTA GAC TCA AGC GGT CAA GCC AAA5276 Ile Asp Asp Lys Gly Val Ser Phe Val Asp Ser Ser Gly Gln Ala Lys1510 1515 1520 GCA AAC ACC CCT GTG CTA AGT GCC AAT GGG CTG GAC CTG GGTGGC AAG 5324 Ala Asn Thr Pro Val Leu Ser Ala Asn Gly Leu Asp Leu Gly GlyLys 1525 1530 1535 GTC ATC AGT AAT GTG GGC AAA GGC ACA AAA GAT ACC GACGCT GCC AAT 5372 Val Ile Ser Asn Val Gly Lys Gly Thr Lys Asp Thr Asp AlaAla Asn 1540 1545 1550 1555 GTA CAA CAG TTA AAC GAA GTA CGC AAC TTG TTGGGT CTT GGT AAT GCT 5420 Val Gln Gln Leu Asn Glu Val Arg Asn Leu Leu GlyLeu Gly Asn Ala 1560 1565 1570 GGT AAT GAT AAC GCT GAC GGC AAT CAG GTAAAC ATT GCC GAC ATC AAA 5468 Gly Asn Asp Asn Ala Asp Gly Asn Gln Val AsnIle Ala Asp Ile Lys 1575 1580 1585 AAA GAC CCA AAT TCA GGT TCA TCA TCTAAC CGC ACT GTC ATC AAA GCA 5516 Lys Asp Pro Asn Ser Gly Ser Ser Ser AsnArg Thr Val Ile Lys Ala 1590 1595 1600 GGC ACG GTA CTT GGC GGT AAA GGTAAT AAC GAT ACC GAA AAA CTT GCC 5564 Gly Thr Val Leu Gly Gly Lys Gly AsnAsn Asp Thr Glu Lys Leu Ala 1605 1610 1615 ACT GGT GGT ATA CAA GTG GGCGTG GAT AAA GAC GGC AAC GCT AAC GGC 5612 Thr Gly Gly Ile Gln Val Gly ValAsp Lys Asp Gly Asn Ala Asn Gly 1620 1625 1630 1635 GAT TTA AGC AAT GTTTGG GTC AAA ACC CAA AAA GAT GGC AGC AAA AAA 5660 Asp Leu Ser Asn Val TrpVal Lys Thr Gln Lys Asp Gly Ser Lys Lys 1640 1645 1650 GCC CTG CTC GCCACT TAT AAC GCC GCA GGT CAG ACC AAC TAT TTG ACC 5708 Ala Leu Leu Ala ThrTyr Asn Ala Ala Gly Gln Thr Asn Tyr Leu Thr 1655 1660 1665 AAC AAC CCCGCA GAA GCC ATT GAC AGA ATA AAT GAA CAA GGT ATC CGC 5756 Asn Asn Pro AlaGlu Ala Ile Asp Arg Ile Asn Glu Gln Gly Ile Arg 1670 1675 1680 TTC TTCCAT GTC AAC GAT GGC AAT CAA GAG CCT GTG GTA CAA GGG CGT 5804 Phe Phe HisVal Asn Asp Gly Asn Gln Glu Pro Val Val Gln Gly Arg 1685 1690 1695 AACGGC ATT GAC TCA AGT GCC TCA GGC AAG CAC TCA GTG GCG ATA GGT 5852 Asn GlyIle Asp Ser Ser Ala Ser Gly Lys His Ser Val Ala Ile Gly 1700 1705 17101715 TTC CAG GCC AAG GCA GAT GGT GAA GCC GCC GTT GCC ATA GGC AGA CAA5900 Phe Gln Ala Lys Ala Asp Gly Glu Ala Ala Val Ala Ile Gly Arg Gln1720 1725 1730 ACC CAA GCA GGC AAC CAA TCC ATC GCC ATC GGT GAT AAC GCACAA GCC 5948 Thr Gln Ala Gly Asn Gln Ser Ile Ala Ile Gly Asp Asn Ala GlnAla 1735 1740 1745 ACG GGC GAT CAA TCC ATC GCC ATC GGT ACA GGC AAT GTGGTA GCA GGT 5996 Thr Gly Asp Gln Ser Ile Ala Ile Gly Thr Gly Asn Val ValAla Gly 1750 1755 1760 AAG CAC TCT GGT GCC ATC GGC GAC CCA AGC ACT GTTAAG GCT GAT AAC 6044 Lys His Ser Gly Ala Ile Gly Asp Pro Ser Thr Val LysAla Asp Asn 1765 1770 1775 AGT TAC AGT GTG GGT AAT AAC AAC CAG TTT ACCGAT GCC ACT CAA ACC 6092 Ser Tyr Ser Val Gly Asn Asn Asn Gln Phe Thr AspAla Thr Gln Thr 1780 1785 1790 1795 GAT GTC TTT GGT GTG GGC AAT AAC ATCACC GTG ACC GAA AGT AAC TCG 6140 Asp Val Phe Gly Val Gly Asn Asn Ile ThrVal Thr Glu Ser Asn Ser 1800 1805 1810 GTT GCC TTA GGT TCA AAC TCT GCCATC AGT GCA GGC ACA CAC GCA GGC 6188 Val Ala Leu Gly Ser Asn Ser Ala IleSer Ala Gly Thr His Ala Gly 1815 1820 1825 ACA CAA GCC AAA AAA TCT GACGGC ACA GCA GGT ACA ACC ACC ACA GCA 6236 Thr Gln Ala Lys Lys Ser Asp GlyThr Ala Gly Thr Thr Thr Thr Ala 1830 1835 1840 GGT GCA ACC GGT ACG GTTAAA GGC TTT GCT GGA CAA ACG GCG GTT GGT 6284 Gly Ala Thr Gly Thr Val LysGly Phe Ala Gly Gln Thr Ala Val Gly 1845 1850 1855 GCG GTC TCC GTG GGTGCC TCA GGT GCT GAA CGC CGT ATC CAA AAT GTG 6332 Ala Val Ser Val Gly AlaSer Gly Ala Glu Arg Arg Ile Gln Asn Val 1860 1865 1870 1875 GCA GCA GGTGAG GTC AGT GCC ACC AGC ACC GAT GCG GTC AAT GGT AGC 6380 Ala Ala Gly GluVal Ser Ala Thr Ser Thr Asp Ala Val Asn Gly Ser 1880 1885 1890 CAG TTGTAC AAA GCC ACC CAA AGC ATT GCC AAC GCA ACC AAT GAG CTT 6428 Gln Leu TyrLys Ala Thr Gln Ser Ile Ala Asn Ala Thr Asn Glu Leu 1895 1900 1905 GACCAT CGT ATC CAC CAA AAC GAA AAT AAG GCC AAT GCA GGG ATT TCA 6476 Asp HisArg Ile His Gln Asn Glu Asn Lys Ala Asn Ala Gly Ile Ser 1910 1915 1920TCA GCG ATG GCG ATG GCG TCC ATG CCA CAA GCC TAC ATT CCT GGC AGA 6524 SerAla Met Ala Met Ala Ser Met Pro Gln Ala Tyr Ile Pro Gly Arg 1925 19301935 TCC ATG GTT ACC GGG GGT ATT GCC ACC CAC AAC GGT CAA GGT GCG GTG6572 Ser Met Val Thr Gly Gly Ile Ala Thr His Asn Gly Gln Gly Ala Val1940 1945 1950 1955 GCA GTG GGA CTG TCG AAG CTG TCG GAT AAT GGT CAA TGGGTA TTT AAA 6620 Ala Val Gly Leu Ser Lys Leu Ser Asp Asn Gly Gln Trp ValPhe Lys 1960 1965 1970 ATC AAT GGT TCA GCC GAT ACC CAA GGC CAT GTA GGGGCG GCA GTT GGT 6668 Ile Asn Gly Ser Ala Asp Thr Gln Gly His Val Gly AlaAla Val Gly 1975 1980 1985 GCA GGT TTT CAC TTT TAAGCCATAA ATCGCAAGATTTTACTTAAA AATCAATCTC 6723 Ala Gly Phe His Phe 1990 ACCATAGTTGTATAAAACAG CATCAGCATC AGTCATATTA CTGATGCTGA TGTTTTTTAT 6783 CACTTAAACCATTTTACCGC TCAAGTGATT CTCTTTCACC ATGACCAAAT CGCCATTGAT 6843 CATAGGTAAACTTATTGAGT AAATTTTATC AATGTAGTTG TTAGATATGG TTAAAATTGT 6903 GCCATTGACCAAAAAATGAC CGATTTATCC CGAAAATTTC TGATTATGAT CCGTTGACCT 6963 GCAGGTCGAC6973 5976 base pairs nucleic acid single linear 2 GTGATCGGTG CAACGCTCAGTGGCAGTGCT TATGCTCAAA AAAAAGATAC CAAACATATC 60 GCAATTGGTG AACAAAACCAGCCAAGACGC TCAGGCACTG CCAAGGCGGA CGGTGATCGA 120 GCCATTGCTA TTGGTGAAAATGCTAACGCA CAGGGCGGTC AAGCCATCGC CATCGGTAGT 180 AGTAATAAAA CTGTCAATGGAAGCAGTTTG GATAAGATAG GTACCGATGC TACGGGTCAA 240 GAGTCCATCG CCATCGGTGGTGATGTAAAG GCTAGTGGTG ATGCCTCGAT TGCCATCGGT 300 AGTGATGACT TACATTTGCTTGATCAGCAT GGTAATCCTA AACATCCGAA AGGTACTCTG 360 ATTAACGATC TTATTAACGGCCATGCAGTA TTAAAAGAAA TACGAAGCTC AAAGGATAAT 420 GATGTAAAAT ATAGACGCACAACCGCAAGC GGACACGCCA GTACTGCAGT GGGAGCCATG 480 TCATATGCAC AGGGTCATTTTTCCAACGCC TTTGGTACAC GGGCAACAGC TAAAAGTGCC 540 TATTCCTTGG CAGTGGGTCTTGCCGCCACA GCCGAGGGCC AATCTACAAT CGCTATTGGT 600 TCTGATGCAA CATCTAGCTCGTTGGGAGCG ATAGCCCTTG GTGCAGGTAC TCGTGCTCAG 660 CTACAGGGCA GTATTGCCCTAGGTCAAGGT TCTGTTGTCA CTCAGAGTGA TAATAATTCT 720 AGACCGGCCT ATACACCAAATACCCAGGCA CTAGACCCCA AGTTTCAAGC CACCAATAAT 780 ACGAAGGCGG GTCCACTTTCCATTGGTAGT AACTCTATCA AACGTAAAAT CATCAATGTC 840 GGTGCAGGTG TTAATAAAACCGATGCGGTC AATGTGGCAC AGCTAGAAGC GGTGGTGAAG 900 TGGGCTAAGG AGCGTAGAATTACTTTTCAG GGTGATGATA ACAGTACTGA CGTAAAAATA 960 GGTTTGGATA ATACTTTAACTATTAAAGGT GGTGCAGAGA CCAACGCATT AACCGATAAT 1020 AATATCGGTG TGGTAAAAGAGGCTGATAAT AGTGGTCTGA AAGTTAAACT TGCTAAAACT 1080 TTAAACAATC TTACTGAGGTGAATACAACT ACATTAAATG CCACAACCAC AGTTAAGGTA 1140 GGTAGTAGTA GTAGTACTACAGCTGAATTA TTGAGTGATA GTTTAACCTT TACCCAGCCC 1200 AATACAGGCA GTCAAAGCACAAGCAAAACC GTCTATGGCG TTAATGGGGT GAAGTTTACT 1260 AATAATGCAG AAACAACAGCAGCAATCGGC ACTACTCGTA TTACCAGAGA TAAAATTGGC 1320 TTTGCTCGAG ATGGTGATGTTGATGAAAAA CAAGCACCAT ATTTGGATAA AAAACAACTT 1380 AAAGTGGGTA GTGTTGCAATTACCATAGAC AATGGCATTG ATGCAGGTAA TAAAAAGATC 1440 AGTAATCTTG CCAAAGGTAGCAGTGCTAAC GATGCGGTTA CCATCGAACA GCTCAAAGCC 1500 GCCAAGCCTA CTTTAAACGCAGGCGCTGGC ATCAGTGTCA CACCTACTGA AATATCAGTT 1560 GATGCTAAGA GTGGCAATGTTACCGCCCCA ACTTACAACA TTGGCGTGAA AACCACCGAG 1620 CTTAACAGTG ATGGCACTAGTGATAAATTT AGTGTTAAGG GTAGTGGTAC GAACAATAGC 1680 TTAGTTACCG CCGAACATTTGGCAAGCTAT CTAAATGAAG TCAATCGAAC GGCTGACAGT 1740 GCTCTACAAA GCTTTACCGTTAAAGAAGAA GACGATGATG ACGCCAACGC TATCACCGTG 1800 GCTAAAGATA CGACAAAAAATGCCGGCGCA GTCAGCATCT TAAAACTCAA AGGTAAAAAC 1860 GGTCTAACGG TTGCTACCAAAAAAGATGGT ACGGTTACCT TTGGGCTTAG CCAAGATAGC 1920 GGTCTGACCA TTGGCAAAAGCACCCTAAAC AACGATGGCT TGACTGTTAA AGATACCAAC 1980 GAACAAATCC AAGTCGGTGCTAATGGCATT AAATTTACTA ATGTGAATGG TAGTAATCCA 2040 GGTACTGGCA TTGCAAATACCGCTCGCATT ACCAGAGATA AAATTGGCTT TGCTGGTTCT 2100 GATGGTGCAG TTGATACAAACAAACCTTAT CTTGATCAAG ACAAGCTACA AGTTGGCAAT 2160 GTTAAGATTA CCAACACTGGCATTAACGCA GGTGGTAAAG CCATCACAGG GCTGTCCCCA 2220 ACACTGCCTA GCATTGCCGATCAAAGTAGC CGCAACATAG AACTGGGCAA TACAATCCAA 2280 GACAAAGACA AATCCAACGCTGCCAGCATT AATGATATAT TAAATACAGG CTTTAACCTA 2340 AAAAATAATA ACAACCCCATTGACTTTGTC TCCACTTATG ACATTGTTGA CTTTGCCAAT 2400 GGCAATGCCA CCACCGCCACAGTAACCCAT GATACCGCTA ACAAAACCAG TAAAGTGGTA 2460 TATGATGTGA ATGTGGATGATACAACCATT CATCTAACAG GCACTGATGA CAATAAAAAA 2520 CTTGGCGTCA AAACCACCAAACTGAACAAA ACAAGTGCTA ATGGTAATAC AGCAACTAAC 2580 TTTAATGTTA ACTCTAGTGATGAAGATGCC CTTGTTAACG CCAAAGACAT CGCCGAAAAT 2640 CTAAACACCC TAGCCAAGGAAATTCACACC ACCAAAGGCA CAGCAGACAC CGCCCTACAA 2700 ACCTTTACCG TTAAAAAGGTAGATGAAAAT AATAATGCTG ATGACGCCAA CGCCATCACC 2760 GTGGGTCAAA AGAACGCAAATAATCAAGTC AACACCCTAA CACTCAAAGG TGAAAACGGT 2820 CTTAATATTA AAACCGACAAAAATGGTACG GTTACCTTTG GCATTAACAC CACAAGCGGT 2880 CTTAAAGCCG GCAAAAGCACCCTAAACGAC GGTGGCTTGT CTATTAAAAA CCCCACTGGT 2940 AGCGAACAAA TCCAAGTCGGTGCTGATGGC GTGAAGTTTG CCAAGGTTAA TAATAATGGT 3000 GTTGTAGGTG CTGGCATTGATGGCACAACT CGCATTACCA GAGATGAAAT TGGCTTTACT 3060 GGGACTAATG GCTCACTTGATAAAAGCAAA CCCCACCTAA GCAAAGACGG CATTAACGCA 3120 GGTGGTAAAA AGATTACCAACATTCAATCA GGTGAGATTG CCCAAAACAG CCATGATGCT 3180 GTGACAGGCG GCAAGATTTATGATTTAAAA ACCGAACTTG AAAACAAAAT CAGCAGTACT 3240 GCCAAAACAG CACAAAACTCATTACACGAA TTCTCAGTAG CAGATGAACA AGGTAATAAC 3300 TTTACGGTTA GTAACCCTTACTCCAGTTAT GACACCTCAA AGACCTCTGA TGTCATCACC 3360 TTTGCAGGTG AAAACGGCATTACCACCAAG GTAAATAAAG GTGTGGTGCG TGTGGGCATT 3420 GACCAAACCA AAGGCTTAACCACGCCTAAG CTGACCGTGG GTAATAATAA TGGCAAAGGC 3480 ATTGTCATTG ACAGCCAAAATGGTCAAAAT ACCATCACAG GACTAAGCAA CACTCTAGCT 3540 AATGTTACCA ATGATAAAGGTAGCGTACGC ACCACAGAAC AGGGCAATAT AATCAAAGAC 3600 GAAGACAAAA CCCGTGCCGCCAGCATTGTT GATGTGCTAA GCGCAGGCTT TAACTTGCAA 3660 GGCAATGGTG AAGCGGTTGACTTTGTCTCC ACTTATGACA CCGTCAACTT TGCCGATGGC 3720 AATGCCACCA CCGCTAAGGTGACCTATGAT GACACAAGCA AAACCAGTAA AGTGGTCTAT 3780 GATGTCAATG TGGATGATACAACCATTGAA GTTAAAGATA AAAAACTTGG CGTAAAAACC 3840 ACCACATTGA CCAGTACTGGCACAGGTGCT AATAAATTTG CCCTAAGCAA TCAAGCTACT 3900 GGCGATGCGC TTGTCAAGGCCAGTGATATC GTTGCTCATC TAAACACCTT ATCTGGCGAC 3960 ATCCAAACTG CCAAAGGGGCAAGCCAAGCG AACAACTCAG CAGGCTATGT GGATGCTGAT 4020 GGCAATAAGG TCATCTATGACAGTACCGAT AACAAGTACT ATCAAGCCAA AAATGATGGC 4080 ACAGTTGATA AAACCAAAGAAGTTGCCAAA GACAAACTGG TCGCCCAAGC CCAAACCCCA 4140 GATGGCACAT TGGCTCAAATGAATGTCAAA TCAGTCATTA ACAAAGAACA AGTAAATGAT 4200 GCCAATAAAA AGCAAGGCATCAATGAAGAC AACGCCTTTG TTAAAGGACT TGAAAAAGCC 4260 GCTTCTGATA ACAAAACCAAAAACGCCGCA GTAACTGTGG GTGATTTAAA TGCCGTTGCC 4320 CAAACACCGC TGACCTTTGCAGGGGATACA GGCACAACGG CTAAAAAACT GGGCGAGACT 4380 TTGACCATCA AAGGTGGGCAAACAGACACC AATAAGCTAA CCGATAATAA CATCGGTGTG 4440 GTAGCAGGTA CTGATGGCTTCACTGTCAAA CTTGCCAAAG ACCTAACCAA TCTTAACAGC 4500 GTTAATGCAG GTGGCACCAAAATTGATGAC AAAGGCGTGT CTTTTGTAGA CTCAAGCGGT 4560 CAAGCCAAAG CAAACACCCCTGTGCTAAGT GCCAATGGGC TGGACCTGGG TGGCAAGGTC 4620 ATCAGTAATG TGGGCAAAGGCACAAAAGAT ACCGACGCTG CCAATGTACA ACAGTTAAAC 4680 GAAGTACGCA ACTTGTTGGGTCTTGGTAAT GCTGGTAATG ATAACGCTGA CGGCAATCAG 4740 GTAAACATTG CCGACATCAAAAAAGACCCA AATTCAGGTT CATCATCTAA CCGCACTGTC 4800 ATCAAAGCAG GCACGGTACTTGGCGGTAAA GGTAATAACG ATACCGAAAA ACTTGCCACT 4860 GGTGGTATAC AAGTGGGCGTGGATAAAGAC GGCAACGCTA ACGGCGATTT AAGCAATGTT 4920 TGGGTCAAAA CCCAAAAAGATGGCAGCAAA AAAGCCCTGC TCGCCACTTA TAACGCCGCA 4980 GGTCAGACCA ACTATTTGACCAACAACCCC GCAGAAGCCA TTGACAGAAT AAATGAACAA 5040 GGTATCCGCT TCTTCCATGTCAACGATGGC AATCAAGAGC CTGTGGTACA AGGGCGTAAC 5100 GGCATTGACT CAAGTGCCTCAGGCAAGCAC TCAGTGGCGA TAGGTTTCCA GGCCAAGGCA 5160 GATGGTGAAG CCGCCGTTGCCATAGGCAGA CAAACCCAAG CAGGCAACCA ATCCATCGCC 5220 ATCGGTGATA ACGCACAAGCCACGGGCGAT CAATCCATCG CCATCGGTAC AGGCAATGTG 5280 GTAGCAGGTA AGCACTCTGGTGCCATCGGC GACCCAAGCA CTGTTAAGGC TGATAACAGT 5340 TACAGTGTGG GTAATAACAACCAGTTTACC GATGCCACTC AAACCGATGT CTTTGGTGTG 5400 GGCAATAACA TCACCGTGACCGAAAGTAAC TCGGTTGCCT TAGGTTCAAA CTCTGCCATC 5460 AGTGCAGGCA CACACGCAGGCACACAAGCC AAAAAATCTG ACGGCACAGC AGGTACAACC 5520 ACCACAGCAG GTGCAACCGGTACGGTTAAA GGCTTTGCTG GACAAACGGC GGTTGGTGCG 5580 GTCTCCGTGG GTGCCTCAGGTGCTGAACGC CGTATCCAAA ATGTGGCAGC AGGTGAGGTC 5640 AGTGCCACCA GCACCGATGCGGTCAATGGT AGCCAGTTGT ACAAAGCCAC CCAAAGCATT 5700 GCCAACGCAA CCAATGAGCTTGACCATCGT ATCCACCAAA ACGAAAATAA GGCCAATGCA 5760 GGGATTTCAT CAGCGATGGCGATGGCGTCC ATGCCACAAG CCTACATTCC TGGCAGATCC 5820 ATGGTTACCG GGGGTATTGCCACCCACAAC GGTCAAGGTG CGGTGGCAGT GGGACTGTCG 5880 AAGCTGTCGG ATAATGGTCAATGGGTATTT AAAATCAATG GTTCAGCCGA TACCCAAGGC 5940 CATGTAGGGG CGGCAGTTGGTGCAGGTTTT CACTTT 5976 1992 amino acids amino acid single linear 3 MetIle Gly Ala Thr Leu Ser Gly Ser Ala Tyr Ala Gln Lys Lys Asp 1 5 10 15Thr Lys His Ile Ala Ile Gly Glu Gln Asn Gln Pro Arg Arg Ser Gly 20 25 30Thr Ala Lys Ala Asp Gly Asp Arg Ala Ile Ala Ile Gly Glu Asn Ala 35 40 45Asn Ala Gln Gly Gly Gln Ala Ile Ala Ile Gly Ser Ser Asn Lys Thr 50 55 60Val Asn Gly Ser Ser Leu Asp Lys Ile Gly Thr Asp Ala Thr Gly Gln 65 70 7580 Glu Ser Ile Ala Ile Gly Gly Asp Val Lys Ala Ser Gly Asp Ala Ser 85 9095 Ile Ala Ile Gly Ser Asp Asp Leu His Leu Leu Asp Gln His Gly Asn 100105 110 Pro Lys His Pro Lys Gly Thr Leu Ile Asn Asp Leu Ile Asn Gly His115 120 125 Ala Val Leu Lys Glu Ile Arg Ser Ser Lys Asp Asn Asp Val LysTyr 130 135 140 Arg Arg Thr Thr Ala Ser Gly His Ala Ser Thr Ala Val GlyAla Met 145 150 155 160 Ser Tyr Ala Gln Gly His Phe Ser Asn Ala Phe GlyThr Arg Ala Thr 165 170 175 Ala Lys Ser Ala Tyr Ser Leu Ala Val Gly LeuAla Ala Thr Ala Glu 180 185 190 Gly Gln Ser Thr Ile Ala Ile Gly Ser AspAla Thr Ser Ser Ser Leu 195 200 205 Gly Ala Ile Ala Leu Gly Ala Gly ThrArg Ala Gln Leu Gln Gly Ser 210 215 220 Ile Ala Leu Gly Gln Gly Ser ValVal Thr Gln Ser Asp Asn Asn Ser 225 230 235 240 Arg Pro Ala Tyr Thr ProAsn Thr Gln Ala Leu Asp Pro Lys Phe Gln 245 250 255 Ala Thr Asn Asn ThrLys Ala Gly Pro Leu Ser Ile Gly Ser Asn Ser 260 265 270 Ile Lys Arg LysIle Ile Asn Val Gly Ala Gly Val Asn Lys Thr Asp 275 280 285 Ala Val AsnVal Ala Gln Leu Glu Ala Val Val Lys Trp Ala Lys Glu 290 295 300 Arg ArgIle Thr Phe Gln Gly Asp Asp Asn Ser Thr Asp Val Lys Ile 305 310 315 320Gly Leu Asp Asn Thr Leu Thr Ile Lys Gly Gly Ala Glu Thr Asn Ala 325 330335 Leu Thr Asp Asn Asn Ile Gly Val Val Lys Glu Ala Asp Asn Ser Gly 340345 350 Leu Lys Val Lys Leu Ala Lys Thr Leu Asn Asn Leu Thr Glu Val Asn355 360 365 Thr Thr Thr Leu Asn Ala Thr Thr Thr Val Lys Val Gly Ser SerSer 370 375 380 Ser Thr Thr Ala Glu Leu Leu Ser Asp Ser Leu Thr Phe ThrGln Pro 385 390 395 400 Asn Thr Gly Ser Gln Ser Thr Ser Lys Thr Val TyrGly Val Asn Gly 405 410 415 Val Lys Phe Thr Asn Asn Ala Glu Thr Thr AlaAla Ile Gly Thr Thr 420 425 430 Arg Ile Thr Arg Asp Lys Ile Gly Phe AlaArg Asp Gly Asp Val Asp 435 440 445 Glu Lys Gln Ala Pro Tyr Leu Asp LysLys Gln Leu Lys Val Gly Ser 450 455 460 Val Ala Ile Thr Ile Asp Asn GlyIle Asp Ala Gly Asn Lys Lys Ile 465 470 475 480 Ser Asn Leu Ala Lys GlySer Ser Ala Asn Asp Ala Val Thr Ile Glu 485 490 495 Gln Leu Lys Ala AlaLys Pro Thr Leu Asn Ala Gly Ala Gly Ile Ser 500 505 510 Val Thr Pro ThrGlu Ile Ser Val Asp Ala Lys Ser Gly Asn Val Thr 515 520 525 Ala Pro ThrTyr Asn Ile Gly Val Lys Thr Thr Glu Leu Asn Ser Asp 530 535 540 Gly ThrSer Asp Lys Phe Ser Val Lys Gly Ser Gly Thr Asn Asn Ser 545 550 555 560Leu Val Thr Ala Glu His Leu Ala Ser Tyr Leu Asn Glu Val Asn Arg 565 570575 Thr Ala Asp Ser Ala Leu Gln Ser Phe Thr Val Lys Glu Glu Asp Asp 580585 590 Asp Asp Ala Asn Ala Ile Thr Val Ala Lys Asp Thr Thr Lys Asn Ala595 600 605 Gly Ala Val Ser Ile Leu Lys Leu Lys Gly Lys Asn Gly Leu ThrVal 610 615 620 Ala Thr Lys Lys Asp Gly Thr Val Thr Phe Gly Leu Ser GlnAsp Ser 625 630 635 640 Gly Leu Thr Ile Gly Lys Ser Thr Leu Asn Asn AspGly Leu Thr Val 645 650 655 Lys Asp Thr Asn Glu Gln Ile Gln Val Gly AlaAsn Gly Ile Lys Phe 660 665 670 Thr Asn Val Asn Gly Ser Asn Pro Gly ThrGly Ile Ala Asn Thr Ala 675 680 685 Arg Ile Thr Arg Asp Lys Ile Gly PheAla Gly Ser Asp Gly Ala Val 690 695 700 Asp Thr Asn Lys Pro Tyr Leu AspGln Asp Lys Leu Gln Val Gly Asn 705 710 715 720 Val Lys Ile Thr Asn ThrGly Ile Asn Ala Gly Gly Lys Ala Ile Thr 725 730 735 Gly Leu Ser Pro ThrLeu Pro Ser Ile Ala Asp Gln Ser Ser Arg Asn 740 745 750 Ile Glu Leu GlyAsn Thr Ile Gln Asp Lys Asp Lys Ser Asn Ala Ala 755 760 765 Ser Ile AsnAsp Ile Leu Asn Thr Gly Phe Asn Leu Lys Asn Asn Asn 770 775 780 Asn ProIle Asp Phe Val Ser Thr Tyr Asp Ile Val Asp Phe Ala Asn 785 790 795 800Gly Asn Ala Thr Thr Ala Thr Val Thr His Asp Thr Ala Asn Lys Thr 805 810815 Ser Lys Val Val Tyr Asp Val Asn Val Asp Asp Thr Thr Ile His Leu 820825 830 Thr Gly Thr Asp Asp Asn Lys Lys Leu Gly Val Lys Thr Thr Lys Leu835 840 845 Asn Lys Thr Ser Ala Asn Gly Asn Thr Ala Thr Asn Phe Asn ValAsn 850 855 860 Ser Ser Asp Glu Asp Ala Leu Val Asn Ala Lys Asp Ile AlaGlu Asn 865 870 875 880 Leu Asn Thr Leu Ala Lys Glu Ile His Thr Thr LysGly Thr Ala Asp 885 890 895 Thr Ala Leu Gln Thr Phe Thr Val Lys Lys ValAsp Glu Asn Asn Asn 900 905 910 Ala Asp Asp Ala Asn Ala Ile Thr Val GlyGln Lys Asn Ala Asn Asn 915 920 925 Gln Val Asn Thr Leu Thr Leu Lys GlyGlu Asn Gly Leu Asn Ile Lys 930 935 940 Thr Asp Lys Asn Gly Thr Val ThrPhe Gly Ile Asn Thr Thr Ser Gly 945 950 955 960 Leu Lys Ala Gly Lys SerThr Leu Asn Asp Gly Gly Leu Ser Ile Lys 965 970 975 Asn Pro Thr Gly SerGlu Gln Ile Gln Val Gly Ala Asp Gly Val Lys 980 985 990 Phe Ala Lys ValAsn Asn Asn Gly Val Val Gly Ala Gly Ile Asp Gly 995 1000 1005 Thr ThrArg Ile Thr Arg Asp Glu Ile Gly Phe Thr Gly Thr Asn Gly 1010 1015 1020Ser Leu Asp Lys Ser Lys Pro His Leu Ser Lys Asp Gly Ile Asn Ala 10251030 1035 1040 Gly Gly Lys Lys Ile Thr Asn Ile Gln Ser Gly Glu Ile AlaGln Asn 1045 1050 1055 Ser His Asp Ala Val Thr Gly Gly Lys Ile Tyr AspLeu Lys Thr Glu 1060 1065 1070 Leu Glu Asn Lys Ile Ser Ser Thr Ala LysThr Ala Gln Asn Ser Leu 1075 1080 1085 His Glu Phe Ser Val Ala Asp GluGln Gly Asn Asn Phe Thr Val Ser 1090 1095 1100 Asn Pro Tyr Ser Ser TyrAsp Thr Ser Lys Thr Ser Asp Val Ile Thr 1105 1110 1115 1120 Phe Ala GlyGlu Asn Gly Ile Thr Thr Lys Val Asn Lys Gly Val Val 1125 1130 1135 ArgVal Gly Ile Asp Gln Thr Lys Gly Leu Thr Thr Pro Lys Leu Thr 1140 11451150 Val Gly Asn Asn Asn Gly Lys Gly Ile Val Ile Asp Ser Gln Asn Gly1155 1160 1165 Gln Asn Thr Ile Thr Gly Leu Ser Asn Thr Leu Ala Asn ValThr Asn 1170 1175 1180 Asp Lys Gly Ser Val Arg Thr Thr Glu Gln Gly AsnIle Ile Lys Asp 1185 1190 1195 1200 Glu Asp Lys Thr Arg Ala Ala Ser IleVal Asp Val Leu Ser Ala Gly 1205 1210 1215 Phe Asn Leu Gln Gly Asn GlyGlu Ala Val Asp Phe Val Ser Thr Tyr 1220 1225 1230 Asp Thr Val Asn PheAla Asp Gly Asn Ala Thr Thr Ala Lys Val Thr 1235 1240 1245 Tyr Asp AspThr Ser Lys Thr Ser Lys Val Val Tyr Asp Val Asn Val 1250 1255 1260 AspAsp Thr Thr Ile Glu Val Lys Asp Lys Lys Leu Gly Val Lys Thr 1265 12701275 1280 Thr Thr Leu Thr Ser Thr Gly Thr Gly Ala Asn Lys Phe Ala LeuSer 1285 1290 1295 Asn Gln Ala Thr Gly Asp Ala Leu Val Lys Ala Ser AspIle Val Ala 1300 1305 1310 His Leu Asn Thr Leu Ser Gly Asp Ile Gln ThrAla Lys Gly Ala Ser 1315 1320 1325 Gln Ala Asn Asn Ser Ala Gly Tyr ValAsp Ala Asp Gly Asn Lys Val 1330 1335 1340 Ile Tyr Asp Ser Thr Asp AsnLys Tyr Tyr Gln Ala Lys Asn Asp Gly 1345 1350 1355 1360 Thr Val Asp LysThr Lys Glu Val Ala Lys Asp Lys Leu Val Ala Gln 1365 1370 1375 Ala GlnThr Pro Asp Gly Thr Leu Ala Gln Met Asn Val Lys Ser Val 1380 1385 1390Ile Asn Lys Glu Gln Val Asn Asp Ala Asn Lys Lys Gln Gly Ile Asn 13951400 1405 Glu Asp Asn Ala Phe Val Lys Gly Leu Glu Lys Ala Ala Ser AspAsn 1410 1415 1420 Lys Thr Lys Asn Ala Ala Val Thr Val Gly Asp Leu AsnAla Val Ala 1425 1430 1435 1440 Gln Thr Pro Leu Thr Phe Ala Gly Asp ThrGly Thr Thr Ala Lys Lys 1445 1450 1455 Leu Gly Glu Thr Leu Thr Ile LysGly Gly Gln Thr Asp Thr Asn Lys 1460 1465 1470 Leu Thr Asp Asn Asn IleGly Val Val Ala Gly Thr Asp Gly Phe Thr 1475 1480 1485 Val Lys Leu AlaLys Asp Leu Thr Asn Leu Asn Ser Val Asn Ala Gly 1490 1495 1500 Gly ThrLys Ile Asp Asp Lys Gly Val Ser Phe Val Asp Ser Ser Gly 1505 1510 15151520 Gln Ala Lys Ala Asn Thr Pro Val Leu Ser Ala Asn Gly Leu Asp Leu1525 1530 1535 Gly Gly Lys Val Ile Ser Asn Val Gly Lys Gly Thr Lys AspThr Asp 1540 1545 1550 Ala Ala Asn Val Gln Gln Leu Asn Glu Val Arg AsnLeu Leu Gly Leu 1555 1560 1565 Gly Asn Ala Gly Asn Asp Asn Ala Asp GlyAsn Gln Val Asn Ile Ala 1570 1575 1580 Asp Ile Lys Lys Asp Pro Asn SerGly Ser Ser Ser Asn Arg Thr Val 1585 1590 1595 1600 Ile Lys Ala Gly ThrVal Leu Gly Gly Lys Gly Asn Asn Asp Thr Glu 1605 1610 1615 Lys Leu AlaThr Gly Gly Ile Gln Val Gly Val Asp Lys Asp Gly Asn 1620 1625 1630 AlaAsn Gly Asp Leu Ser Asn Val Trp Val Lys Thr Gln Lys Asp Gly 1635 16401645 Ser Lys Lys Ala Leu Leu Ala Thr Tyr Asn Ala Ala Gly Gln Thr Asn1650 1655 1660 Tyr Leu Thr Asn Asn Pro Ala Glu Ala Ile Asp Arg Ile AsnGlu Gln 1665 1670 1675 1680 Gly Ile Arg Phe Phe His Val Asn Asp Gly AsnGln Glu Pro Val Val 1685 1690 1695 Gln Gly Arg Asn Gly Ile Asp Ser SerAla Ser Gly Lys His Ser Val 1700 1705 1710 Ala Ile Gly Phe Gln Ala LysAla Asp Gly Glu Ala Ala Val Ala Ile 1715 1720 1725 Gly Arg Gln Thr GlnAla Gly Asn Gln Ser Ile Ala Ile Gly Asp Asn 1730 1735 1740 Ala Gln AlaThr Gly Asp Gln Ser Ile Ala Ile Gly Thr Gly Asn Val 1745 1750 1755 1760Val Ala Gly Lys His Ser Gly Ala Ile Gly Asp Pro Ser Thr Val Lys 17651770 1775 Ala Asp Asn Ser Tyr Ser Val Gly Asn Asn Asn Gln Phe Thr AspAla 1780 1785 1790 Thr Gln Thr Asp Val Phe Gly Val Gly Asn Asn Ile ThrVal Thr Glu 1795 1800 1805 Ser Asn Ser Val Ala Leu Gly Ser Asn Ser AlaIle Ser Ala Gly Thr 1810 1815 1820 His Ala Gly Thr Gln Ala Lys Lys SerAsp Gly Thr Ala Gly Thr Thr 1825 1830 1835 1840 Thr Thr Ala Gly Ala ThrGly Thr Val Lys Gly Phe Ala Gly Gln Thr 1845 1850 1855 Ala Val Gly AlaVal Ser Val Gly Ala Ser Gly Ala Glu Arg Arg Ile 1860 1865 1870 Gln AsnVal Ala Ala Gly Glu Val Ser Ala Thr Ser Thr Asp Ala Val 1875 1880 1885Asn Gly Ser Gln Leu Tyr Lys Ala Thr Gln Ser Ile Ala Asn Ala Thr 18901895 1900 Asn Glu Leu Asp His Arg Ile His Gln Asn Glu Asn Lys Ala AsnAla 1905 1910 1915 1920 Gly Ile Ser Ser Ala Met Ala Met Ala Ser Met ProGln Ala Tyr Ile 1925 1930 1935 Pro Gly Arg Ser Met Val Thr Gly Gly IleAla Thr His Asn Gly Gln 1940 1945 1950 Gly Ala Val Ala Val Gly Leu SerLys Leu Ser Asp Asn Gly Gln Trp 1955 1960 1965 Val Phe Lys Ile Asn GlySer Ala Asp Thr Gln Gly His Val Gly Ala 1970 1975 1980 Ala Val Gly AlaGly Phe His Phe 1985 1990 1833 amino acids amino acid single linear 4Met Ser Tyr Ala Gln Gly His Phe Ser Asn Ala Phe Gly Thr Arg Ala 1 5 1015 Thr Ala Lys Ser Ala Tyr Ser Leu Ala Val Gly Leu Ala Ala Thr Ala 20 2530 Glu Gly Gln Ser Thr Ile Ala Ile Gly Ser Asp Ala Thr Ser Ser Ser 35 4045 Leu Gly Ala Ile Ala Leu Gly Ala Gly Thr Arg Ala Gln Leu Gln Gly 50 5560 Ser Ile Ala Leu Gly Gln Gly Ser Val Val Thr Gln Ser Asp Asn Asn 65 7075 80 Ser Arg Pro Ala Tyr Thr Pro Asn Thr Gln Ala Leu Asp Pro Lys Phe 8590 95 Gln Ala Thr Asn Asn Thr Lys Ala Gly Pro Leu Ser Ile Gly Ser Asn100 105 110 Ser Ile Lys Arg Lys Ile Ile Asn Val Gly Ala Gly Val Asn LysThr 115 120 125 Asp Ala Val Asn Val Ala Gln Leu Glu Ala Val Val Lys TrpAla Lys 130 135 140 Glu Arg Arg Ile Thr Phe Gln Gly Asp Asp Asn Ser ThrAsp Val Lys 145 150 155 160 Ile Gly Leu Asp Asn Thr Leu Thr Ile Lys GlyGly Ala Glu Thr Asn 165 170 175 Ala Leu Thr Asp Asn Asn Ile Gly Val ValLys Glu Ala Asp Asn Ser 180 185 190 Gly Leu Lys Val Lys Leu Ala Lys ThrLeu Asn Asn Leu Thr Glu Val 195 200 205 Asn Thr Thr Thr Leu Asn Ala ThrThr Thr Val Lys Val Gly Ser Ser 210 215 220 Ser Ser Thr Thr Ala Glu LeuLeu Ser Asp Ser Leu Thr Phe Thr Gln 225 230 235 240 Pro Asn Thr Gly SerGln Ser Thr Ser Lys Thr Val Tyr Gly Val Asn 245 250 255 Gly Val Lys PheThr Asn Asn Ala Glu Thr Thr Ala Ala Ile Gly Thr 260 265 270 Thr Arg IleThr Arg Asp Lys Ile Gly Phe Ala Arg Asp Gly Asp Val 275 280 285 Asp GluLys Gln Ala Pro Tyr Leu Asp Lys Lys Gln Leu Lys Val Gly 290 295 300 SerVal Ala Ile Thr Ile Asp Asn Gly Ile Asp Ala Gly Asn Lys Lys 305 310 315320 Ile Ser Asn Leu Ala Lys Gly Ser Ser Ala Asn Asp Ala Val Thr Ile 325330 335 Glu Gln Leu Lys Ala Ala Lys Pro Thr Leu Asn Ala Gly Ala Gly Ile340 345 350 Ser Val Thr Pro Thr Glu Ile Ser Val Asp Ala Lys Ser Gly AsnVal 355 360 365 Thr Ala Pro Thr Tyr Asn Ile Gly Val Lys Thr Thr Glu LeuAsn Ser 370 375 380 Asp Gly Thr Ser Asp Lys Phe Ser Val Lys Gly Ser GlyThr Asn Asn 385 390 395 400 Ser Leu Val Thr Ala Glu His Leu Ala Ser TyrLeu Asn Glu Val Asn 405 410 415 Arg Thr Ala Asp Ser Ala Leu Gln Ser PheThr Val Lys Glu Glu Asp 420 425 430 Asp Asp Asp Ala Asn Ala Ile Thr ValAla Lys Asp Thr Thr Lys Asn 435 440 445 Ala Gly Ala Val Ser Ile Leu LysLeu Lys Gly Lys Asn Gly Leu Thr 450 455 460 Val Ala Thr Lys Lys Asp GlyThr Val Thr Phe Gly Leu Ser Gln Asp 465 470 475 480 Ser Gly Leu Thr IleGly Lys Ser Thr Leu Asn Asn Asp Gly Leu Thr 485 490 495 Val Lys Asp ThrAsn Glu Gln Ile Gln Val Gly Ala Asn Gly Ile Lys 500 505 510 Phe Thr AsnVal Asn Gly Ser Asn Pro Gly Thr Gly Ile Ala Asn Thr 515 520 525 Ala ArgIle Thr Arg Asp Lys Ile Gly Phe Ala Gly Ser Asp Gly Ala 530 535 540 ValAsp Thr Asn Lys Pro Tyr Leu Asp Gln Asp Lys Leu Gln Val Gly 545 550 555560 Asn Val Lys Ile Thr Asn Thr Gly Ile Asn Ala Gly Gly Lys Ala Ile 565570 575 Thr Gly Leu Ser Pro Thr Leu Pro Ser Ile Ala Asp Gln Ser Ser Arg580 585 590 Asn Ile Glu Leu Gly Asn Thr Ile Gln Asp Lys Asp Lys Ser AsnAla 595 600 605 Ala Ser Ile Asn Asp Ile Leu Asn Thr Gly Phe Asn Leu LysAsn Asn 610 615 620 Asn Asn Pro Ile Asp Phe Val Ser Thr Tyr Asp Ile ValAsp Phe Ala 625 630 635 640 Asn Gly Asn Ala Thr Thr Ala Thr Val Thr HisAsp Thr Ala Asn Lys 645 650 655 Thr Ser Lys Val Val Tyr Asp Val Asn ValAsp Asp Thr Thr Ile His 660 665 670 Leu Thr Gly Thr Asp Asp Asn Lys LysLeu Gly Val Lys Thr Thr Lys 675 680 685 Leu Asn Lys Thr Ser Ala Asn GlyAsn Thr Ala Thr Asn Phe Asn Val 690 695 700 Asn Ser Ser Asp Glu Asp AlaLeu Val Asn Ala Lys Asp Ile Ala Glu 705 710 715 720 Asn Leu Asn Thr LeuAla Lys Glu Ile His Thr Thr Lys Gly Thr Ala 725 730 735 Asp Thr Ala LeuGln Thr Phe Thr Val Lys Lys Val Asp Glu Asn Asn 740 745 750 Asn Ala AspAsp Ala Asn Ala Ile Thr Val Gly Gln Lys Asn Ala Asn 755 760 765 Asn GlnVal Asn Thr Leu Thr Leu Lys Gly Glu Asn Gly Leu Asn Ile 770 775 780 LysThr Asp Lys Asn Gly Thr Val Thr Phe Gly Ile Asn Thr Thr Ser 785 790 795800 Gly Leu Lys Ala Gly Lys Ser Thr Leu Asn Asp Gly Gly Leu Ser Ile 805810 815 Lys Asn Pro Thr Gly Ser Glu Gln Ile Gln Val Gly Ala Asp Gly Val820 825 830 Lys Phe Ala Lys Val Asn Asn Asn Gly Val Val Gly Ala Gly IleAsp 835 840 845 Gly Thr Thr Arg Ile Thr Arg Asp Glu Ile Gly Phe Thr GlyThr Asn 850 855 860 Gly Ser Leu Asp Lys Ser Lys Pro His Leu Ser Lys AspGly Ile Asn 865 870 875 880 Ala Gly Gly Lys Lys Ile Thr Asn Ile Gln SerGly Glu Ile Ala Gln 885 890 895 Asn Ser His Asp Ala Val Thr Gly Gly LysIle Tyr Asp Leu Lys Thr 900 905 910 Glu Leu Glu Asn Lys Ile Ser Ser ThrAla Lys Thr Ala Gln Asn Ser 915 920 925 Leu His Glu Phe Ser Val Ala AspGlu Gln Gly Asn Asn Phe Thr Val 930 935 940 Ser Asn Pro Tyr Ser Ser TyrAsp Thr Ser Lys Thr Ser Asp Val Ile 945 950 955 960 Thr Phe Ala Gly GluAsn Gly Ile Thr Thr Lys Val Asn Lys Gly Val 965 970 975 Val Arg Val GlyIle Asp Gln Thr Lys Gly Leu Thr Thr Pro Lys Leu 980 985 990 Thr Val GlyAsn Asn Asn Gly Lys Gly Ile Val Ile Asp Ser Gln Asn 995 1000 1005 GlyGln Asn Thr Ile Thr Gly Leu Ser Asn Thr Leu Ala Asn Val Thr 1010 10151020 Asn Asp Lys Gly Ser Val Arg Thr Thr Glu Gln Gly Asn Ile Ile Lys1025 1030 1035 1040 Asp Glu Asp Lys Thr Arg Ala Ala Ser Ile Val Asp ValLeu Ser Ala 1045 1050 1055 Gly Phe Asn Leu Gln Gly Asn Gly Glu Ala ValAsp Phe Val Ser Thr 1060 1065 1070 Tyr Asp Thr Val Asn Phe Ala Asp GlyAsn Ala Thr Thr Ala Lys Val 1075 1080 1085 Thr Tyr Asp Asp Thr Ser LysThr Ser Lys Val Val Tyr Asp Val Asn 1090 1095 1100 Val Asp Asp Thr ThrIle Glu Val Lys Asp Lys Lys Leu Gly Val Lys 1105 1110 1115 1120 Thr ThrThr Leu Thr Ser Thr Gly Thr Gly Ala Asn Lys Phe Ala Leu 1125 1130 1135Ser Asn Gln Ala Thr Gly Asp Ala Leu Val Lys Ala Ser Asp Ile Val 11401145 1150 Ala His Leu Asn Thr Leu Ser Gly Asp Ile Gln Thr Ala Lys GlyAla 1155 1160 1165 Ser Gln Ala Asn Asn Ser Ala Gly Tyr Val Asp Ala AspGly Asn Lys 1170 1175 1180 Val Ile Tyr Asp Ser Thr Asp Asn Lys Tyr TyrGln Ala Lys Asn Asp 1185 1190 1195 1200 Gly Thr Val Asp Lys Thr Lys GluVal Ala Lys Asp Lys Leu Val Ala 1205 1210 1215 Gln Ala Gln Thr Pro AspGly Thr Leu Ala Gln Met Asn Val Lys Ser 1220 1225 1230 Val Ile Asn LysGlu Gln Val Asn Asp Ala Asn Lys Lys Gln Gly Ile 1235 1240 1245 Asn GluAsp Asn Ala Phe Val Lys Gly Leu Glu Lys Ala Ala Ser Asp 1250 1255 1260Asn Lys Thr Lys Asn Ala Ala Val Thr Val Gly Asp Leu Asn Ala Val 12651270 1275 1280 Ala Gln Thr Pro Leu Thr Phe Ala Gly Asp Thr Gly Thr ThrAla Lys 1285 1290 1295 Lys Leu Gly Glu Thr Leu Thr Ile Lys Gly Gly GlnThr Asp Thr Asn 1300 1305 1310 Lys Leu Thr Asp Asn Asn Ile Gly Val ValAla Gly Thr Asp Gly Phe 1315 1320 1325 Thr Val Lys Leu Ala Lys Asp LeuThr Asn Leu Asn Ser Val Asn Ala 1330 1335 1340 Gly Gly Thr Lys Ile AspAsp Lys Gly Val Ser Phe Val Asp Ser Ser 1345 1350 1355 1360 Gly Gln AlaLys Ala Asn Thr Pro Val Leu Ser Ala Asn Gly Leu Asp 1365 1370 1375 LeuGly Gly Lys Val Ile Ser Asn Val Gly Lys Gly Thr Lys Asp Thr 1380 13851390 Asp Ala Ala Asn Val Gln Gln Leu Asn Glu Val Arg Asn Leu Leu Gly1395 1400 1405 Leu Gly Asn Ala Gly Asn Asp Asn Ala Asp Gly Asn Gln ValAsn Ile 1410 1415 1420 Ala Asp Ile Lys Lys Asp Pro Asn Ser Gly Ser SerSer Asn Arg Thr 1425 1430 1435 1440 Val Ile Lys Ala Gly Thr Val Leu GlyGly Lys Gly Asn Asn Asp Thr 1445 1450 1455 Glu Lys Leu Ala Thr Gly GlyIle Gln Val Gly Val Asp Lys Asp Gly 1460 1465 1470 Asn Ala Asn Gly AspLeu Ser Asn Val Trp Val Lys Thr Gln Lys Asp 1475 1480 1485 Gly Ser LysLys Ala Leu Leu Ala Thr Tyr Asn Ala Ala Gly Gln Thr 1490 1495 1500 AsnTyr Leu Thr Asn Asn Pro Ala Glu Ala Ile Asp Arg Ile Asn Glu 1505 15101515 1520 Gln Gly Ile Arg Phe Phe His Val Asn Asp Gly Asn Gln Glu ProVal 1525 1530 1535 Val Gln Gly Arg Asn Gly Ile Asp Ser Ser Ala Ser GlyLys His Ser 1540 1545 1550 Val Ala Ile Gly Phe Gln Ala Lys Ala Asp GlyGlu Ala Ala Val Ala 1555 1560 1565 Ile Gly Arg Gln Thr Gln Ala Gly AsnGln Ser Ile Ala Ile Gly Asp 1570 1575 1580 Asn Ala Gln Ala Thr Gly AspGln Ser Ile Ala Ile Gly Thr Gly Asn 1585 1590 1595 1600 Val Val Ala GlyLys His Ser Gly Ala Ile Gly Asp Pro Ser Thr Val 1605 1610 1615 Lys AlaAsp Asn Ser Tyr Ser Val Gly Asn Asn Asn Gln Phe Thr Asp 1620 1625 1630Ala Thr Gln Thr Asp Val Phe Gly Val Gly Asn Asn Ile Thr Val Thr 16351640 1645 Glu Ser Asn Ser Val Ala Leu Gly Ser Asn Ser Ala Ile Ser AlaGly 1650 1655 1660 Thr His Ala Gly Thr Gln Ala Lys Lys Ser Asp Gly ThrAla Gly Thr 1665 1670 1675 1680 Thr Thr Thr Ala Gly Ala Thr Gly Thr ValLys Gly Phe Ala Gly Gln 1685 1690 1695 Thr Ala Val Gly Ala Val Ser ValGly Ala Ser Gly Ala Glu Arg Arg 1700 1705 1710 Ile Gln Asn Val Ala AlaGly Glu Val Ser Ala Thr Ser Thr Asp Ala 1715 1720 1725 Val Asn Gly SerGln Leu Tyr Lys Ala Thr Gln Ser Ile Ala Asn Ala 1730 1735 1740 Thr AsnGlu Leu Asp His Arg Ile His Gln Asn Glu Asn Lys Ala Asn 1745 1750 17551760 Ala Gly Ile Ser Ser Ala Met Ala Met Ala Ser Met Pro Gln Ala Tyr1765 1770 1775 Ile Pro Gly Arg Ser Met Val Thr Gly Gly Ile Ala Thr HisAsn Gly 1780 1785 1790 Gln Gly Ala Val Ala Val Gly Leu Ser Lys Leu SerAsp Asn Gly Gln 1795 1800 1805 Trp Val Phe Lys Ile Asn Gly Ser Ala AspThr Gln Gly His Val Gly 1810 1815 1820 Ala Ala Val Gly Ala Gly Phe HisPhe 1825 1830 19 amino acids amino acid single linear 5 Asn Val Lys SerVal Ile Asn Lys Glu Gln Val Asn Asp Ala Asn Lys 1 5 10 15 Gln Gly Ile 16amino acids amino acid single linear 6 Asn Val Lys Ser Val Ile Asn LysGlu Gln Val Asn Asp Ala Asn Lys 1 5 10 15 60 base pairs nucleic acidsingle linear 7 AATGTCAAAT CAGTCATTAA CAAAGAACAA GTAAATGATG CCAATAAAAAGCAAGGCATC 60 20 amino acids amino acid single linear 8 Asn Val Lys SerVal Ile Asn Lys Glu Gln Val Asn Asp Ala Asn Lys 1 5 10 15 Lys Gln GlyIle 20 30 amino acids amino acid single linear 9 Met Ile Gly Ala Thr LeuSer Gly Ser Ala Tyr Ala Gln Lys Lys Asp 1 5 10 15 Thr Lys His Ile AlaIle Gly Glu Gln Asn Gln Pro Arg Arg 20 25 30 30 amino acids amino acidsingle linear 10 Ser Gly Thr Ala Lys Ala Asp Gly Asp Arg Ala Ile Ala IleGly Glu 1 5 10 15 Asn Ala Asn Ala Gln Gly Gly Gln Ala Ile Ala Ile GlySer 20 25 30

What we claim is:
 1. An isolated and purified outer membrane protein ofa Moraxella catarrhalis strain having an apparent molecular mass ofabout 200 kDa, as determined by SDS-PAGE, characterized by an amino acidsequence selected from the group consisting of: (a) an amino acidsequence containing SEQ ID NO:3; (b) an amino acid sequence encoded bySEQ ID NO:1 or 2; (c) an amino acid sequence containing SEQ ID NO:8; (d)an amino acid sequence containing an amino acid sequence encoded by SEQID NO:7; (e) an amino acid sequence containing SEQ ID NO:6; (f) an aminoacid sequence containing SEQ ID NO:9; and (g) an amino acid sequencecontaining SEQ ID NO:10.
 2. The protein of claim 1 which is at leastabout 70 wt % pure.
 3. The protein of claim 2 which is at least about 95wt % pure.
 4. The protein of claim 1 in the form of an aqueous solutionthereof.
 5. The protein of claim 1 having the amino acid composition asshown in Table III.
 6. A peptide consisting of an amino acid sequenceselected from the group consisting of SEQ ID NO:9 and SEQ ID NO:10.